Fungal Spore Richness and Abundance of Allergenic Taxa: Comparing a Portable Impactor and Passive Trap Indoors and Outdoors in an Urban Setting.

Fungal spores are common airborne allergens, and fungal richness has been implicated in allergic disease. Amplicon sequencing of environmental DNA from air samples is a promising method to estimate fungal spore richness with semi-quantification of hundreds of taxa and can be combined with quantitative PCR to derive abundance estimates. However, it remains unclear how the choice of air sampling method influences these estimates. This study compared active sampling with a portable impactor and passive sampling with a passive trap over different durations to estimate fungal spore richness and the abundance of allergenic taxa. Air sampling was conducted indoors and outdoors at 12 residences, including repeated measurements with a portable impactor and passive traps with 1-day and 7-day durations. ITS2 amplicon sequence data were transformed to spore equivalents estimated by quantitative PCR, repeated active samples were combined, and abundance-based rarefaction was performed to standardize sample coverage for estimation of genus-level richness and spore abundance. Rarefied fungal richness was similar between methods indoors but higher for passive traps with a 7-day duration outdoors. Rarefied abundance of allergenic genera was similar between methods but some genera had lower abundance for passive traps with a 1-day duration, which differed indoors and outdoors indicating stochasticity in the collection of spores on collocated samplers. This study found that similar estimates of fungal spore richness and abundance of allergenic taxa can be obtained using a portable impactor or a passive trap within one day and that increased passive sample duration provides limited additional information.

Fungal Spore Richness in School Classrooms is Related to Surrounding Forest in a Season-Dependent Manner.

Airborne fungal spores are important aeroallergens that are remarkably diverse in terms of taxonomic richness. Indoor fungal richness is dominated by outdoor fungi and is geographically patterned, but the influence of natural landscape is unclear. We aimed to elucidate the relationship between indoor fungal spore richness and natural landscape by examining the amount of surrounding forest cover. Passive sampling of airborne fungal spores was conducted in 24 schools in Taiwan during hot and cool seasons, and amplicon sequencing was used to study fungal spore (genus) richness targeting the internal transcribed spacer 2 (ITS2) region. In total, 693 fungal genera were identified, 12 of which were ubiquitous. Despite overall similarity of fungal spore richness between seasons, Basidiomycota and Ascomycota richness increased during the hot and cool seasons, respectively. Fungal spore richness in schools had a strong positive correlation with the amount of surrounding forest cover during the cool season, but not during the hot season. Fungal assemblages in schools were more similar during the hot season due to the increased ubiquity of Agaricomycetes genera. These observations indicate dispersal limitation at the kilometer scale during the cool season and increased long-distance dispersal during the hot season. Several allergenic fungi were commonly identified in schools, including some previously overlooked by conventional methods, which may be targeted as sensitizing agents in future investigations into atopic conditions. More generally, the relative importance of fungal spore richness in the development, chronicity, and severity of atopic conditions in children requires investigation.

Dynamic changes in genetic diversity, drug resistance mutations, and treatment outcomes of falciparum malaria from the low-transmission to the pre-elimination phase on the islands of São Tomé and Príncipe.

BACKGROUND: With effective vector control and case management, substantial progress has been made towards eliminating malaria on the islands of São Tomé and Príncipe (STP). This study assessed the dynamic changes in the genetic diversity of Plasmodium falciparum, the anti-malarial drug resistance mutations, and malaria treatment outcomes between 2010 and 2016 to provide insights for the prevention of malaria rebounding. METHODS: Polymorphic regions of merozoite surface proteins 1 and 2 (msp1 and msp2) were sequenced in 118 dried blood spots (DBSs) collected from malaria patients who had visited the Central Hospital in 2010-2016. Mutations in the multi-drug resistance I (pfmdr1), chloroquine resistance transporter (pfcrt), and kelch 13 (pfk13) genes were analysed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing in 111 DBSs. A total of 7482 cases that completed a 28-day follow-up were evaluated for treatment outcomes based on the microscopic results. Regression models were used to characterize factors associated with levels of parasite density and treatment failures. RESULTS: Parasite strains in STP showed significant changes during and after the peak incidence in 2012. The prevalent allelic type in msp1 changed from K1 to MAD20, and that in msp2 changed from 3D7/IC to FC27. The dominant alleles of drug-resistance markers were pfmdr1 86Y, 184F, D1246, and pfcrt 76 T (Y-F-D-T, 51.4%). The average parasite density in malaria cases declined threefold from low-transmission (2010-2013) to pre-elimination period (2014-2016). Logistic regression models showed that patients with younger age (OR for age = 0.97-0.98, p < 0.001), higher initial parasite density (log10-transformed, OR = 1.44, p < 0.001), and receiving quinine treatment (compared to artemisinin-based combination therapy, OR = 1.91-1.96, p < 0.001) were more likely to experience treatment failures during follow-up. CONCLUSIONS: Plasmodium falciparum in STP had experienced changes in prevalent strains, and increased mutation frequencies in drug-resistance genes from the low-transmission to the pre-elimination settings. Notably, patients with younger age and receiving quinine treatment were more likely to show parasitological treatment failure during follow-up. Therapeutic efficacy should be carefully monitored to inform future treatment policy in STP.

Effects of indoor residual spraying and outdoor larval control on Anopheles coluzzii from São Tomé and Príncipe, two islands with pre-eliminated malaria.

BACKGROUND: Vector control is a key component of malaria prevention. Two major vector control strategies have been implemented in São Tomé and Príncipe (STP), indoor residual spraying (IRS) and outdoor larval control using Bacillus thuringiensis israelensis (Bti). This study evaluated post-intervention effects of control strategies on vector population density, composition, and knockdown resistance mutation, and their implications for malaria epidemiology in STP. METHODS: Mosquitoes were collected by indoor and outdoor human landing catches and mosquito light traps in seven districts. Mosquito density was calculated by numbers of captured adult mosquitoes/house/working hour. Mitochondrial cytochrome c oxidase subunit I (COI) was PCR amplified and sequenced to understand the spatial-temporal population composition of malaria vector in STP. Knockdown resistance L1014F mutation was detected using allele-specific PCR. To estimate the malaria transmission risks in STP, a negative binomial regression model was constructed. The response variable was monthly incidence, and the explanatory variables were area, rainfall, entomological inoculation rate (EIR), and kdr mutation frequency. RESULTS: Malaria vector in STP is exophilic Anopheles coluzzii with significant population differentiation between Príncipe and São Tomé (mean FST = 0.16, p < 0.001). Both vector genetic diversity and knockdown resistance mutation were relatively low in Príncipe (mean of kdr frequency = 15.82%) compared to São Tomé (mean of kdr frequency = 44.77%). Annual malaria incidence rate in STP had been rapidly controlled from 37 to 2.1% by three rounds of country-wide IRS from 2004 to 2007. Long-term application of Bti since 2007 kept the mosquito density under 10 mosquitoes/house/hr/month, and malaria incidence rate under 5% after 2008, except for a rising that occurred in 2012 (incidence rate = 6.9%). Risk factors of area (São Tomé compared to Príncipe), rainfall, outdoor EIR, and kdr mutation frequency could significantly increase malaria incidence by 9.33-11.50, 1.25, 1.07, and 1.06 fold, respectively. CONCLUSIONS: Indoor residual spraying could rapidly decrease Anopheles density and malaria incidence in STP. Outdoor larval control using Bti is a sustainable approach for controlling local vector with exophilic feature and insecticide resistance problem. Vector control interventions should be intensified especially at the north-eastern part of São Tomé to minimize impacts of outbreaks.

Concatenated ScaA and TSA56 Surface Antigen Sequences Reflect Genome-Scale Phylogeny of Orientia tsutsugamushi: An Analysis Including Two Genomes from Taiwan.

Orientia tsutsugamushi is an obligate intracellular bacterium associated with trombiculid mites and is the causative agent of scrub typhus, a life-threatening febrile disease. Strain typing of O. tsutsugamushi is based on its immunodominant surface antigen, 56-kDa type-specific antigen (TSA56). However, TSA56 gene sequence-based phylogenetic analysis is only partially congruent with core genome-based phylogenetic analysis. Thus, this study investigated whether concatenated surface antigen sequences, including surface cell antigen (Sca) proteins, can reflect the genome-scale phylogeny of O. tsutsugamushi. Complete genomes were obtained for two common O. tsutsugamushi strains in Taiwan, TW-1 and TW-22, and the core genome/proteome was identified for 11 O. tsutsugamushi strains. Phylogenetic analysis was performed using maximum likelihood (ML) and neighbor-joining (NJ) methods, and the congruence between trees was assessed using a quartet similarity measure. Phylogenetic analysis based on 691 concatenated core protein sequences produced identical tree topologies with ML and NJ methods. Among TSA56 and core Sca proteins (ScaA, ScaC, ScaD, and ScaE), TSA56 trees were most similar to the core protein tree, and ScaA trees were the least similar. However, concatenated ScaA and TSA56 sequences produced trees that were highly similar to the core protein tree, the NJ tree being more similar. Strain-level characterization of O. tsutsugamushi may be improved by coanalyzing ScaA and TSA56 sequences, which are also important targets for their combined immunogenicity.

Infestation prevalence, spatio-temporal distribution, phylogenetic positioning, and pathogen investigation of Argas persicus ticks in domestic hens (Gallus gallus domesticus) from Pakistan.

Soft ticks pose significant health risks as vectors of various pathogens. This study explored the spatio-temporal distribution and genetic relationships of the soft tick species Argas persicus infesting domestic hens (Gallus gallus domesticus) across different districts in Pakistan. An examination of 778 hens revealed a notable tick infestation prevalence of 70.82%, with a total of 1299 ticks collected from 551 hens. The overall mean intensity was 2.19 soft ticks per infested chicken, and the overall mean abundance was 1.61 soft ticks per examined hen. Morphological identification confirmed all collected ticks (n = 1210) as A. persicus, comprising 719 males, 333 females, 121 nymphs, and 38 larvae. The Haveli, Muzaffarabad, and Kotli districts had the highest infestation rates, while Bagh had the lowest. Molecular analyses of tick DNA, focusing on 16S rDNA and 12S rDNA sequences, revealed genetic similarities among A. persicus soft ticks from Pakistan and other regions, providing insights into their evolutionary history. Importantly, no Babesia, Rickettsia, or Anaplasma infections were detected in the examined samples. These findings enhance the understanding of soft tick infestation patterns and the genetic diversity of A. persicus in the studied region.

Nationwide Inventory of Mosquitoes and the Distribution of Invasive Aedes (Stegomyia) albopictus (Skuse, 1894) on the Islands of Sao Tome and Principe in Central Africa.

Aedes albopictus (Skuse, 1894), a mosquito originating in Asia, has been introduced to Africa since the 2000s. The mosquito is not only a nuisance but is capable of transmitting various arboviruses. The current study summarized our entomological surveys in the Democratic Republic of Sao Tome and Principe during 2000 to 2016. Adult mosquitoes were collected by sweep nets, human landing catches, and Centers for Disease Control (CDC) light traps, and the immatures were collected from water-filled habitats at 15 sentinel sites and reared to adulthood. Species identification was performed based on morphologic characteristics. Fragments of the cytochrome C oxidase subunit I (COI) and the Wolbachia surface protein (wsp) genes were amplified for mosquitoes collected in Principe. New records of four mosquito species were reported. Aedes albopictus was identified in 2015. The larvae were found distributed over the nation and were predominately in artificial water-holding containers (488/2698, 18.1%). The highest positive rate was observed in used tires in Príncipe (114/250, 45.6%). Mitochondrial DNA analysis revealed low genetic diversity among the invasive populations, but all tested specimens were superinfected by Wolbachia. The ability of Ae. albopictus to adapt to new environments and its involvement in disease transmission make the surveillance and control of this species particularly important.

First report of Anaplasma spp., Ehrlichia spp., and Rickettsia spp. in Amblyomma gervaisi ticks infesting monitor lizards (Varanus begalensis) of Pakistan.

Ticks pose significant health risks to both wildlife and humans due to their role as vectors for various pathogens. In this study, we investigated tick infestation patterns, tick-associated pathogens, and genetic relationships within the tick species Amblyomma gervaisi, focusing on its prevalence in monitor lizards (Varanus bengalensis) across different districts in Pakistan. We examined 85 monitor lizards and identified an overall mean intensity of 19.59 ticks per infested lizard and an overall mean abundance of 11.98 ticks per examined lizard. All collected ticks (n = 1019) were morphologically identified as A. gervaisi, including 387 males, 258 females, 353 nymphs, and 21 larvae. The highest tick prevalence was observed in the Buner district, followed by Torghar and Shangla, with the lowest prevalence in Chitral. Lizard captures primarily occurred from May to October, correlating with the period of higher tick infestations. Molecular analysis was conducted on tick DNA, revealing genetic similarities among A. gervaisi ticks based on 16S rDNA and ITS2 sequences. Notably, we found the absence of A. gervaisi ITS2 sequences in the NCBI GenBank, highlighting a gap in existing genetic data. Moreover, our study identified the presence of pathogenic microorganisms, including Ehrlichia sp., Candidatus Ehrlichia dumleri, Anaplasma sp., Francisella sp., Rickettsia sp., and Coxiella sp., in these ticks. BLAST analysis revealed significant similarities between these pathogenic sequences and known strains, emphasizing the potential role of these ticks as vectors for zoonotic diseases. Phylogenetic analyses based on nuclear ITS2 and mitochondrial 16S rDNA genes illustrated the genetic relationships of A. gervaisi ticks from Pakistan with other Amblyomma species, providing insights into their evolutionary history. These findings contribute to our understanding of tick infestation patterns, and tick-borne pathogens in monitor lizards, which has implications for wildlife health, zoonotic disease transmission, and future conservation efforts. Further research in this area is crucial for a comprehensive assessment of the risks associated with tick-borne diseases in both wildlife and humans.

Genetic surveillance reveals low, sustained malaria transmission with clonal replacement in Sao Tome and Principe.

Despite efforts to eliminate malaria in Sao Tome and Principe (STP), cases have recently increased. Understanding residual transmission structure is crucial for developing effective elimination strategies. This study collected surveillance data and generated amplicon sequencing data from 980 samples between 2010 and 2016 to examine the genetic structure of the parasite population. The mean multiplicity of infection (MOI) was 1.3, with 11% polyclonal infections, indicating low transmission intensity. Temporal trends of these genetic metrics did not align with incidence rates, suggesting that changes in genetic metrics may not straightforwardly reflect changes in transmission intensity, particularly in low transmission settings where genetic drift and importation have a substantial impact. While 88% of samples were genetically linked, continuous turnover in genetic clusters and changes in drug-resistance haplotypes were observed. Principal component analysis revealed some STP samples were genetically similar to those from Central and West Africa, indicating possible importation. These findings highlight the need to prioritize several interventions such as targeted interventions against transmission hotspots, reactive case detection, and strategies to reduce the introduction of new parasites into this island nation as it approaches elimination. This study also serves as a case study for implementing genetic surveillance in a low transmission setting.

Rickettsia felis is an emerging human pathogen associated with cat fleas: A review of findings in Taiwan.

Rickettsia felis is an emerging rickettsial agent principally associated with cat fleas (Ctenocephalides felis), formerly discovered in 1990. Since then, clinical cases of R. felis infection have been identified globally by specific DNA sequences in patients with undifferentiated febrile illness, including in Taiwan, but such evidence is limited. R. felis rickettsiosis is self-limiting and easily treated with doxycycline, but its diagnosis remains a challenge. Environmental risk factors for R. felis rickettsiosis have yet to be clearly demonstrated, and its transmission biology is incompletely understood. Cat fleas are naturally infected with R. felis at varying rates, and vector competence in the transmission of R. felis has been demonstrated in animal models, including dogs, which may serve as reservoir hosts. In northern Taiwan, despite ∼20% of cat fleas infesting companion animals consistently found to be infected with R. felis, only a few cases of potential R. felis infection have been identified through a retrospective serological investigation, though without molecular confirmation. Ecological studies have identified divergent R. felis-like organisms in different arthropod hosts, but these strains appear to serve as nonpathogenic endosymbionts. Although its association with disease is limited, we believe cat flea-borne R. felis warrants increased recognition in an aging population due to immunosenescence and the proximity of companion animals to the elderly. Adopting a One Health approach involving collaboration and communication between clinicians, veterinarians, public health practitioners, and environmental scientists will improve our knowledge about this neglected pathogen and promote the prevention and control of vector-borne diseases.

Association of SFG Rickettsia massiliae and Candidatus Rickettsia shennongii with Different Hard Ticks Infesting Livestock Hosts.

Ixodid ticks are responsible for the transmission of various intracellular bacteria, such as the Rickettsia species. Little Information is available about the genetic characterization and epidemiology of Rickettsia spp. The current study was designed to assess the tick species infesting various livestock hosts and the associated Rickettsia spp. in Pakistan. Ticks were collected from different livestock hosts (equids, cattle, buffaloes, sheep, goats, and camels); morphologically identified; and screened for the genetic characterization of Rickettsia spp. by the amplification of partial fragments of the gltA, ompA and ompB genes. Altogether, 707 ticks were collected from 373 infested hosts out of 575 observed hosts. The infested hosts comprised 105 cattle, 71 buffaloes, 70 sheep, 60 goats, 34 camels, and 33 equids. The overall occurrence of Rickettsia spp. was 7.6% (25/330) in the tested ticks. Rickettsia DNA was detected in Rhipicephalus haemaphysaloides (9/50, 18.0%), followed by Rhipicephalus turanicus (13/99, 13.1%), Haemaphysalis cornupunctata (1/18, 5.5%), and Rhipicephalus microplus (2/49, 4.1%); however, no rickettsial DNA was detected in Hyalomma anatolicum (71), Hyalomma dromedarii (35), and Haemaphysalis sulcata (8). Two Rickettsia agents were identified based on partial gltA, ompA, and ompB DNA sequences. The Rickettsia species detected in Rh. haemaphysaloides, Rh. turanicus, and Rh. microplus showed 99-100% identity with Rickettsia sp. and Candidatus Rickettsia shennongii, and in the phylogenetic trees clustered with the corresponding Rickettsia spp. The Rickettsia species detected in Rh. haemaphysaloides, Rh. turanicus, Rh. microplus, and Ha. cornupunctata showed 100% identity with R. massiliae, and in the phylogenetic trees it was clustered with the same species. Candidatus R. shennongii was characterized for the first time in Rh. haemaphysaloides, Rh. turanicus, and Rh. microplus. The presence of SFG Rickettsia spp., including the human pathogen R. massiliae, indicates a zoonotic risk in the study region, thus stressing the need for regular surveillance.

First report on detection of Hepatozoon ayorgbor in Rhipicephalus haemaphysaloides and Hepatozoon colubri in Haemaphysalis sulcata and Hyalomma anatolicum: risks of spillover of Hepatozoon spp. from wildlife to domestic animals.

This study aimed to detect Hepatozoon spp. in ticks infesting asymptomatic domestic animals and to provide insight into their potential spillover from wild to domestic animals. In total, 537 tick specimens were collected in Khyber Pakhtunkhwa, Pakistan, and morphologically identified. The most prevalent tick species was Haemaphysalis cornupunctata (69; 12.8%), followed by Haemaphysalis kashmirensis (62; 11.5%), Rhipicephalus microplus (58; 10.8%), Haemaphysalis montgomeryi (51; 9.5%), Rhipicephalus sanguineus (49; 9.1%), each Haemaphysalis bispinosa and Haemaphysalis sulcata (43; 8.0%), each Hyalomma anatolicum and Rhipicephalus turanicus (37; 6.9%), Rhipicephalus haemaphysaloides (33; 6.1%) Hyalomma scupense (30; 5.6%), and Hyalomma isaaci (25; 4.7%). The extracted DNA from a subset of each tick species was subjected to PCR to amplify 18S rRNA fragments of Hepatozoon spp. By BLAST analysis, the Hepatozoon sp. detected in Hy. anatolicum infesting cows and in Ha. sulcata infesting sheep showed 99.7% maximum identity with Hepatozoon colubri. Similarly, the Hepatozoon sp. detected in R. haemaphysaloides infesting goats shared 99.49% maximum identity with Hepatozoon ayorgbor, and the Hepatozoon sp. detected in R. sanguineus infesting dogs exhibited 99.7% identity with Hepatozoon canis. Having an overall infection rate (9.3%; 16/172), the highest infection rate was recorded for each H. canis, and H. colubri (3.5%; 6/172), followed by H. ayorgbor (2.3%; 4/172). In the phylogenetic tree, H. colubri clustered with corresponding species from Iran, H. ayorgbor clustered with the same species from Croatia, Ghana, and Portugal, and H. canis clustered with the conspecifics from Iran, Israel, Romania, and Zambia. Regarding the potential spillover of Hepatozoon spp. from wildlife through ticks, free ranging animals was at higher risk compared to confined animals (RR = 3.05), animals consuming food from wildlife habitats were at higher risk compared to those consuming domestic food (RR = 3.06), and animals residing in farm buildings located in wildlife habitats were at higher risk compared to those residing in farm buildings located in villages (RR = 3.28). In addition to the first report on H. canis in R. sanguineus in Pakistan, this is the earliest data showing H. ayorgbor in R. haemaphysaloides and H. colubri in Ha. sulcata and Hy. anatolicum. These preliminary findings suggest a potential spillover of Hepatozoon spp. from wild to domestic animals via ticks under certain risk factors.

First Detection of Theileria sinensis-like and Anaplasma capra in Ixodes kashmiricus: With Notes on cox1-Based Phylogenetic Position and New Locality Records.

Ixodes ticks transmit Theileria and Anaplasma species to a wide range of animals. The spreading of ticks and tick-borne pathogens has been attributed to transhumant herds, and research on these uninvestigated issues has been neglected in many countries, including Pakistan. Recently, we used internal transcribed spacer (ITS) and 16S ribosomal DNA partial sequences to genetically characterize Ixodes kashmiricus ticks and their associated Rickettsia spp. However, the data on its cox1 sequence and associated Theileria spp. and Anaplasma spp. are missing. This study aimed to genetically characterize I. kashmiricus based on the cox1 sequence and their associated Theileria spp. and Anaplasma spp. The I. kashmiricus ticks were collected from small ruminants: sheep (Ovis aries) and goats (Capra hircus) of transhumant herds in district Shangla, Dir Upper and Chitral, Khyber Pakhtunkhwa (KP), Pakistan. Out of 129 examined hosts, 94 (72.87%) (56 sheep and 38 goats) were infested by 352 ticks, including adult females (175; 49.7%) followed by nymphs (115; 32.7%) and males (62; 17.6%). For molecular analyses, 121 ticks were subjected to DNA isolation and PCR for the amplification of the cox1 sequence for I. kashmiricus, 18S rDNA for Theileria spp. and 16S rDNA sequences for Anaplasma spp. The obtained cox1 sequence showed 89.29%, 88.78%, and 88.71% identity with Ixodes scapularis, Ixodes gibbosus, and Ixodes apronophorus, respectively. Phylogenetically, the present cox1 sequence clustered with the Ixodes ricinus complex. Additionally, the 18S rDNA sequence showed 98.11% maximum identity with Theileria cf. sinensis and 97.99% identity with Theileria sinensis. Phylogenetically, Theileria spp. clustered with the T. cf. sinensis and T. sinensis. In the case of Anaplasma spp., the 16S rDNA sequence showed 100% identity with Anaplasma capra and phylogenetically clustered with the A. capra. PCR-based DNA detection targeting the amplification of groEL and flaB sequences of Coxiella spp. and Borrelia spp., respectively, was unsuccessful. This is the first phylogenetic report based on cox1 and new locality records of I. kashmiricus, and the associated T. sinensis-like and A. capra. Significant tick surveillance studies are needed in order to determine the epidemiology of Ixodes ticks and their associated pathogens.

Distribution and Risk of Cutaneous Leishmaniasis in Khyber Pakhtunkhwa, Pakistan.

Cutaneous leishmaniasis (CL) is a zoonotic infection caused by obligate intracellular protozoa of the genus Leishmania. This study aimed to investigate CL in Khyber Pakhtunkhwa, Pakistan and to estimate the risk of epidemics. Clinico-epidemiological data of 3188 CL patients were collected from health facilities in 2021. Risk factors were analyzed using the chi-square test. ArcGIS V.10.7.1 was applied for spatial analysis. The association between CL occurrence and climatic variables was examined by Bayesian geostatistical analysis. The clinical data revealed males or individuals younger than 20 years old were more affected. Most patients presented with a single lesion, and the face was the most attacked body part. CL was prevalent in the southern region in winter. A proportional symbol map, a choropleth map, and a digital elevation model map were built to show the distribution of CL. Focal transmission was predicted by inverse distance weighting interpolation. Cluster and outlier analysis identified clusters in Bannu, Dir Lower, and Mardan, and hotspot analysis suggested Bannu as a high-risk foci. Bayesian geostatistical analysis indicated that increasing precipitation and temperature as well as low altitudes were associated with CL infection. The study has provided important information for public health sectors to develop intervention strategies for future CL epidemics.

First record of Alectorobius coniceps (Ixodoidea: Argasidae) and Dermacentor sp. (Ixodoidea: Ixodidae) in Pakistan.

Alectorobius species are soft ticks primarily infesting birds, such as swallows, while Dermacentor species are hard ticks mainly infesting mammals, such as small ruminants. This study for the first time reported on the morphological and molecular bases of two tick species, namely A. coniceps and a Dermacentor sp. in Pakistan. The former species was examined in swallows' nests in Khyber Pakhtunkhwa province, while the latter species was examined in small ruminants in Balochistan province. In total, 25 ticks were collected, with 14 ticks morphologically identified as A. coniceps (males = 9 and females = 5) and 11 ticks identified as Dermacentor sp. (males = 7 and females = 4). Following morphological identification, molecular identification was gained by obtaining 16S rDNA and cox1 sequences for these ticks. The BLAST results for the 16S rDNA and cox1 sequences from A. coniceps shared a maximum identity of 97.46% and 96.49% with the same species from Malta. The BLAST analysis of the 16S rDNA and cox1 sequences from Dermacentor sp. showed maximum identities of 98.42% and 97.45% with Dermacentor pavlovskyi from China. The phylogenetic analysis based on 16S rDNA and cox1 of A. coniceps showed a close evolutionary relationship with the same species. The case of Dermacentor sp., based on 16S DNA and cox1, indicated a close evolutionary relationship with Dermacentor pavlovskyi from China.

First report of spotted fever group Rickettsia aeschlimannii in Hyalomma turanicum, Haemaphysalis bispinosa, and Haemaphysalis montgomeryi infesting domestic animals: updates on the epidemiology of tick-borne Rickettsia aeschlimannii.

Tick-borne Rickettsia spp. have long been known as causative agents for zoonotic diseases. We have previously characterized Rickettsia spp. in different ticks infesting a broad range of hosts in Pakistan; however, knowledge regarding Rickettsia aeschlimannii in Haemaphysalis and Hyalomma ticks is missing. This study aimed to obtain a better understanding about R. aeschlimannii in Pakistan and update the knowledge about its worldwide epidemiology. Among 369 examined domestic animals, 247 (66%) were infested by 872 ticks. Collected ticks were morphologically delineated into three genera, namely, Rhipicephalus, Hyalomma, and Haemaphysalis. Adult females were the most prevalent (number ₌ 376, 43.1%), followed by nymphs (303, 34.74%) and males (193, 22.13%). Overall, genomic DNA samples of 223 tick were isolated and screened for Rickettsia spp. by the amplification of rickettsial gltA, ompA, and ompB partial genes using conventional PCR. Rickettsial DNA was detected in 8 of 223 (3.58%) ticks including nymphs (5 of 122, 4.0%) and adult females (3 of 86, 3.48%). The rickettsial gltA, ompA, and ompB sequences were detected in Hyalomma turanicum (2 nymphs and 1 adult female), Haemaphysalis bispinosa (1 nymph and 1 adult female), and Haemaphysalis montgomeryi (2 nymphs and 1 adult female). These rickettsial sequences showed 99.71-100% identity with R. aeschlimannii and phylogenetically clustered with the same species. None of the tested Rhipicephalus microplus, Hyalomma isaaci, Hyalomma scupense, Rhipicephalus turanicus, Hyalomma anatolicum, Rhipicephalus haemaphysaloides, Rhipicephalus sanguineus, Haemaphysalis cornupunctata, and Haemaphysalis sulcata ticks were found positive for rickettsial DNA. Comprehensive surveillance studies should be adopted to update the knowledge regarding tick-borne zoonotic Rickettsia species, evaluate their risks to humans and livestock, and investigate the unexamined cases of illness after tick bite among livestock holders in the country.

The role of voltage-gated sodium channel genotypes in pyrethroid resistance in Aedes aegypti in Taiwan.

BACKGROUND: Aedes aegypti is the major vector of dengue that threatens public health in tropical and subtropical regions. Pyrethroid-based control strategies effectively control this vector, but the repeated usage of the same insecticides leads to resistance and hampers control efforts. Therefore, efficient and prompt monitoring of insecticide resistance in local mosquito populations is critical for dengue control. METHODOLOGY/PRINCIPAL FINDING: We collected Ae. aegypti in southern Taiwan in March and October 2016. We analyzed the voltage-gated sodium channel (vgsc) genotypes of parentals (G0) and G1 adults after cypermethrin insecticide bioassay. Our results showed that four VGSC mutations (S989P, V1016G, F1534C, and D1763Y) associated with resistance were commonly detected in field-collected Ae. aegypti. The frequencies of these four mutations in the local mosquito population were significantly higher in October (0.29, 0.4, 0.27 and 0.11) than in March (0.09, 0.16, 0.18 and 0.03). Specific vgsc combined genotypes composed of the one to four such mutations (SGFY/SGFY, SVCD/SVCD, SGFY/PGFD, SVCD/SGFY, PGFD/PGFD, and SVCD/PGFD) shifted towards higher frequencies in October, implying their resistance role. In addition, the cypermethrin exposure bioassay data supported the field observations. Moreover, our study observed an association between the resistance level and the proportion of resistance genotypes in the population. CONCLUSIONS/SIGNIFICANCE: This is the first study to demonstrate the role of four-locus vgsc genotypes in resistance evaluation in a local Ae. aegypti population in Taiwan. This alternative method using resistance-associated genotypes as an indicator of practically insecticide resistance monitoring is a useful tool for providing precise and real-time information for decision makers.

Molecular epidemiology of scrub typhus in Taiwan during 2006-2016.

Scrub typhus is the most common endemic vector-borne disease in Taiwan. We identified a total of 4,857 laboratory-confirmed cases during 2006-2016 with hyperendemic foci on offshore islands, including Penghu (778 cases, 16.0%) and Kinmen (716 cases, 14.7%), and eastern Taiwan, including Taitung (628 cases, 12.9%) and Hualien (508 cases, 10.5%). Scrub typhus cases occur year-round throughout Taiwan, with a summer peak in June and July. A total of 545 O. tsutsugamushi isolates were successfully obtained from patients infected in diverse geographic areas, including Taiwan and three offshore islands, and the complete open reading frame of the 56 kDa type-specific antigen gene (tsa56) sequence of these isolates was examined. High phylogenetic diversity was found in these isolates, which could be grouped into 36 distinct sequence types. Most isolates belonged to the Karp (49.9%; 272/545), followed by the TW-22 (17.8%; 97/454) and Kawasaki (14.7%; 80/545) genotypes. In conclusion, our data indicate the widespread presence of tsa56 genotypes closely related to Thailand and Korean strains and the presence of the unique endemic strains TW-12, TW-22, TW-29, and TW-36 in Taiwan.