Complete mitochondrial genome of Dacus vijaysegarani and phylogenetic relationships with congeners and other tephritid fruit flies (Insecta: Diptera).

BACKGROUND: Tephritid fruit flies of the genus Dacus are members of the tribe Dacini, subfamily Dacinae. There are some 274 species worldwide, distributed in Africa and the Asia-Pacific. To date, only five complete mitochondrial genomes (mitogenomes) of Dacus fruit flies have been published and are available in the GenBank. METHODS AND RESULTS: In view of the lack of study on their mitogenome, we sequenced (by next generation sequencing) and annotated the complete mitogenome of D. vijaysegarani from Malaysia to determine its features and phylogenetic relationship. The whole mitogenome of D. vijaysegarani has identical gene order with the published mitogenomes of the genus Dacus, with 13 protein-coding genes, two rRNA genes, 22 tRNAs, a non-coding A + T rich control region, and intergenic spacer and overlap sequences. Phylogenetic analysis based on 15 mitochondrial genes (13 PCGs and two rRNA genes), reveals Dacus, Zeugodacus and Bactrocera forming a distinct clade. The genus Dacus forms a monophyletic group in the subclade containing also the Zeugodacus group; this Dacus-Zeugodacus subclade is distinct from the Bactrocera subclade. D. (Mellesis) vijaysegarani forms a lineage with D. (Mellesis) trimacula in the subcluster containing also the lineage of D. (Mellesis) conopsoides and D. (Callantra) longicornis. D. (Dacus) bivittatus and D. (Didacus) ciliatus form a distinct subcluster. Based on cox1 sequences, the Malaysia and Vietnam taxa of D. vijaysegarani may not be conspecific. CONCLUSIONS: Overall, the mitochondrial genome of D. vijaysegarani provided essential molecular data that could be useful for further studies for species diagnosis, evolution and phylogeny research of other tephritid fruit flies in the future.

Sandwich dot-immunogold filtration assay (DIGFA) for specific immunodiagnosis of active neuroangiostrongyliasis.

Serological tests may yield false-negative results for specific antibodies detection before or at the early seroconversion phase. Tests that detect circulating antigens of Angiostrongylus cantonensis would therefore be of value in diagnosis to distinguish current or past infection. Here, a quick, easy to perform, portable and inexpensive diagnostic device for detection of 31-kDa A. cantonensis specific antigens had been developed. This sandwich dot-immunogold filtration assay (AcDIGFAAg), for detecting active angiostrongyliasis was produced using anti-A. cantonensis polyclonal antibody dotted on the nitrocellulose membrane as a capture agent and colloidal gold-labelled anti-31 kDa A. cantonensis antibody as a detection agent. A well-defined pink dot, indicating positivity, was seen readily by naked eye within 10-15 min. The AcDIGFAAg detected A. cantonensis-specific antigens in cerebrospinal fluid samples from 4 out of 10 serologically confirmed angiostrongyliasis cases and 2 out of 5 suspected cases with negative anti-A. cantonensis antibodies. Among the 19 patient sera with A. cantonensis infection, 2 showed positive reaction by AcDIGFAAg. No positive AcDIGFAAg reaction was observed in all the serum samples with other parasitic diseases, and the healthy controls. The present 'AcDIGFAAg' enables rapid qualitative detection of the specific 31-kDa antigens of A. cantonensis in clinical samples with potential for application even under resource-limited settings.

Evaluation of Rapid IgG4 Test for Diagnosis of Gnathostomiasis.

Human gnathostomiasis is a parasitic disease caused by Gnathostoma nematode infection. A rapid, reliable, and practical immunoassay, named dot immuno-gold filtration assay (DIGFA), was developed to supporting clinical diagnosis of gnathostomiasis. The practical tool detected anti-Gnathostoma-specific IgG4 in human serum using crude extract of third-stage larvae as antigen. The result of the test was shown by anti-human IgG4 monoclonal antibody conjugated colloidal gold. The sensitivity and specificity of the test were both 100% for detection in human sera from patients with gnathostomiasis (13/13) and from healthy negative controls (50/50), respectively. Cross-reactivity with heterogonous serum samples from patients with other helminthiases ranged from 0 (trichinosis, paragonimiasis, clonorchiasis, schistosomiasis, and cysticercosis) to 25.0% (sparganosis), with an average of 6.3% (7/112). Moreover, specific IgG4 antibodies diminished at 6 months after treatment. This study showed that DIGFA for the detection of specific IgG4 in human sera could be a promising tool for the diagnosis of gnathostomiasis and useful for evaluating therapeutic effects.

Differential abundance and core members of the bacterial community associated with wild male Zeugodacus cucurbitae fruit flies (Insecta: Tephritidae) from three geographical regions of Southeast Asia.

Zeugodacus cucurbitae (Coquillet) is one of the most significant and widespread tephritid pest species of agricultural crops. This study reports the bacterial communities associated with Z. cucurbitae from three geographical regions in Southeast Asia (Thailand, Peninsular Malaysia, and Sarawak). The bacterial microbiota were investigated by targeted 16S rRNA gene (V3-V4 region) sequencing using the Illumina Mi-Seq platform. At 97% similarity and filtering at 0.001%, there were seven bacterial phyla and unassigned bacteria, comprising 11 classes, 23 orders, 39 families and 67 genera. The bacterial diversity and richness varied within and among the samples from the three geographical regions. Five phyla were detected for the Sarawak sample, and six each for the Thailand and Peninsular Malaysia samples. Four phyla-Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria-were represented in all the fruit fly specimens, forming the core members of the bacterial community. Proteobacteria was the predominant phylum, followed by Bacteroidetes, Firmicutes, and Actinobacteria. Fifty-three genera were represented in the Thailand sample, 56 in the Peninsular Malaysia sample, and 55 in the Sarawak sample. Forty-two genera were present in all the three geographical regions. The predominant core members were order Enterobacteriales (Proeteobacteria), and family Enterobacteriaceae (Enterobacteriales). Klebsiella (Enterobacteriaceae) was the predominant genus and K. oxytoca the predominant species with all specimens having > 10% relative abundance. The results indicate the presence of a great diversity as well as core members of the bacterial community associated with different populations of Z. cucurbitae.

Differentiating sibling species of Zeugodacus caudatus (Insecta: Tephritidae) by complete mitochondrial genome.

Zeugodacus caudatus is a pest of pumpkin flowers. It has a Palearctic and Oriental distribution. We report here the complete mitochondrial genome of the Malaysian and Indonesian samples of Z. caudatus determined by next-generation sequencing of genomic DNA and determine their taxonomic status as sibling species and phylogeny with other taxa of the genus Zeugodacus. The whole mitogenome of both samples possessed 37 genes (13 protein-coding genes-PCGs, 2 rRNA and 22 tRNA genes) and a control region. The mitogenome of the Indonesian sample (15,885 bp) was longer than that of the Malaysian sample (15,866 bp). In both samples, TΨC-loop was absent in trnF and DHU-loop was absent in trnS1. Molecular phylogeny based on 13 PCGs was concordant with 15 mitochondrial genes (13 PCGs and 2 rRNA genes), with the two samples of Z. caudatus forming a sister group and the genus Zeugodacus was monophyletic. The Malaysian and Indonesian samples of Z. caudatus have a genetic distance of p = 7.8 % based on 13 PCGs and p = 7.0 % based on 15 mitochondrial genes, indicating status of sibling species. They are proposed to be accorded specific status as members of a species complex.

Molecular diagnosis of eosinophilic meningitis due to Angiostrongylus cantonensis (Nematoda: Metastrongyloidea) by polymerase chain reaction-DNA sequencing of cerebrospinal fluids of patients.

Cerebrospinal fluid (CSF) samples from clinically diagnosed patients with detectable Angiostrongylus cantonensis-specific antibodies (n = 10), patients with clinically suspected cases that tested negative for A. cantonensis-antibodies (n = 5) and patients with cerebral gnathostomiasis (n = 2) and neurocysticercosis (n = 2) were examined by a single-step polymerase chain reaction (PCR) method using the AC primers for the 66-kDa native protein gene. The PCR method detected A. cantonensis DNA in CSF samples from four of 10 serologically confirmed angiostrongyliasis cases. The PCR results were negative for the remaining CSF samples. The nucleotide sequences of three positive CSF-PCR samples shared 98.8-99.2% similarity with the reference sequence of A. cantonensis. These results indicate the potential application of this PCR assay with clinical CSF samples for additional support in the confirmation of eosinophilic meningitis due to A. cantonensis.

Rapid Single-Step Immunochromatographic Assay for Angiostrongylus cantonensis Specific Antigen Detection.

Angiostrongylus cantonensis is the major etiological nematode parasite causing eosinophilic meningitis and/or eosinophilic meningoencephalitis in humans. The rapid global spread of Angiostrongylus cantonensis and the emerging occurrence of the infection have exposed the shortcomings of traditional/conventional diagnostics. This has spurred efforts to develop faster, simpler and more scalable platforms that can be decentralized for point-of-need laboratory testing. By far, the point-of-care immunoassays such as the lateral flow assay (LFA) are the best-placed. In this work, a LFA in the form of an immunochromatographic test device (designated AcAgQuickDx), based on the detection of a circulating Angiostrongylus cantonensis-derived antigen, was established using anti-31 kDa Angiostrongylus cantonensis antibody as the capture reagent and anti-Angiostrongylus cantonensis polyclonal antibody as the indicator reagent. The AcAgQuickDx was evaluated for its diagnostic potential with a total of 20 cerebrospinal fluids (CSF) and 105 serum samples from patients with angiostrongyliasis and other clinically related parasitic diseases, as well as serum samples from normal healthy subjects. Three of the ten CSF samples from serologically confirmed angiostrongyliasis cases and two of the five suspected cases with negative anti-Angiostrongylus cantonensis antibodies showed a positive AcAgQuickDx reaction. Likewise, the AcAgQuickDx was able to detect Angiostrongylus cantonensis specific antigens in four serum samples of the 27 serologically confirmed angiostrongyliasis cases. No positive reaction by AcAgQuickDx was observed in any of the CSF (n = 5) and serum (n = 43) samples with other parasitic infections, or the normal healthy controls (n = 35). The AcAgQuickDx enabled the rapid detection of active/acute Angiostrongylus cantonensis infection. It is easy to use, can be transported at room temperature and does not require refrigeration for long-term stability over a wide range of climate. It can supplement existing diagnostic tests for neuroangiostrongyliasis under clinical or field environments, particularly in remote and resource-poor areas.

Immunodiagnostic Detection of Angiostrongylus cantonensis Exposure on Hawaii Island Using Isogeographic 31-kDa Antigen.

Angiostrongylus cantonensis is the leading cause of neuroangiostrongyliasis worldwide, and east Hawaii Island is a hotspot for the disease in the United States. A combination of glycoproteins with molecular weight of 31 kDa has been used as antigen to evaluate antibody response in human serum samples in Thailand with high specificity and sensitivity. In a previous pilot study, the Thailand-isolated 31-kDa proteins showed efficacy in dot-blot tests using serum samples from 435 human volunteers on Hawaii Island. However, we hypothesized that native antigen isolated from Hawaii A. cantonensis may exhibit higher specificity than the Thailand-isolated 31-kDa antigen due to potential minor variation in epitopes between isolates. In this study, 31-kDa glycoproteins were isolated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis from adult A. cantonensis nematodes collected from rats captured on east Hawaii Island. The resultant proteins were purified by electroelution, pooled, bioanalyzed, and quantified. A subset of 148 samples from human participants of the original cohort of 435 was consented for this study, including 12 of the original 15 clinically diagnosed participants. Results of ELISA using the Hawaii-isolated 31-kDa antigen were compared with results of the same serum samples previously tested with both crude Hawaii antigen ELISA and Thailand 31-kDa antigen dot blot. This study shows a seroprevalence in the general population of East Hawaii Island of 25.0%, similar to previous findings of 23.8% seroprevalence in this cohort using crude antigen from Hawaii A. cantonensis and 26.5% using Thailand 31-kDa antigen.

Workshop on research priorities for management and treatment of angiostrongyliasis(1).

In a concluding session of the workshop, the participants developed a list of 115 research and outreach needs, outlining the top 5-7 needs in each of 8 areas (Table). For complete information, including presenter details and abstracts, visit the workshop website at www.hawaii.edu/cowielab/Angio%20website%20home.htm.

Efficacy of plant essential oils for the repellents against chiggers (Leptotrombidium imphalum) vector of scrub typhus.

Scrub typhus caused by the Orientia tsutsugamushi. Rodents, particularly rats, serve as principal reservoir hosts. Infection in man is transmitted by the, chigger bite. Repellents provide an effective agent of protecting individuals from chigger. In the present study 6 plant essential oils were tested for evaluation of their repellent activity against the chigger, Leptotrombidium imphalum. The results showed that Clove oil was significantly more effective than others with ED50 and EC50 of 0.420 mg and 2.3%, followed by Zingiber oil (8.458 mg and 42.3%), Vetiver oil (19.582 mg and 97.9%), Turmeric oil (24.343 mg and 121.7%), Orange oil (27.310 mg and 136.6%) and Boesenbergia oil (30.486 mg and 152.4%). These results suggested that Clove oil was the most efficient repellent against chigger which is the vector for scrub typhus.

Genetic diversity and identity of Ascaris worms from human and pig hosts in Thailand.

Ascaris roundworms are of public health and socio-economic importance worldwide. They are conventionally attributed to two taxa - A. lumbricoides infecting principally human and A. suum infecting principally pig. Phylogenomic analysis has revealed that Ascaris worms from both human and pig are represented in Clades A and B. A recent study indicates that the Ascaris worms from human and pig in Thailand belong to Clade A. We examined adult Ascaris worms from human and pig in Thailand by means of the partial sequences of three mitochondrial genes (cox1, cox2 and nad1) and concatenation of these genes. Phylogenomic analysis indicates that two isolates (H1,H2) of A. lumbricoides from human belonged to Clade B; one isolate (H3) belonged to Clade A (based on cox1, cox2 and concatenated sequences) or as an outlier to Clades A and B (based on nad1 sequences). All the eight isolates of A. suum from pig clustered in Clade A. The partial nad1 and the concatenated sequences revealed two lineages of A. suum isolates which were distinct from the two A. lumbricoides isolates of Clade B. It is evident that greater genetic diversity, and a more robust phylogeny, could be uncovered by the application of multiple genes. In sum, the present study reveals the presence in Thailand of A. lumbricoides from human in Clades A and B which necessitates appropriate treatment and control measures; Clades A and B have been reported to contain haplotypes of Ascaris worms from both human and pig in other parts of the world. A country wide study is needed to elucidate the identity, distribution, prevalence, cross transmission, genetic diversity and phylogeny of the Ascaris worms in Thailand.

Data set on the diversity and core members of bacterial community associated with two specialist fruit flies Bactrocera melastomatos and B. umbrosa (Insecta, Tephritidae).

Bactrocera melastomatos Drew & Hancock and Bactrocera umbrosa (Fabricius) are fruit flies of the subfamily Dacinae under the family Tephritidae [1]. B. melastomatos occurs in India (Andaman Island), Thailand, Peninsular Malaysia, Singapore, and Indonesia (Sumatra, Kalimantan, Java) [1] while B. umbrosa is distributed from southern Thailand and Malaysia to New Guinea and New Caledonia [2]. The adult male flies of B. melastomatos are attracted to Cue lure while the adult male flies of B. umbrosa are attracted to methyl eugenol [3]. Fruit flies of Bactrocera melastomatos infest Melastomataceae while those of B. umbrosa infest Moraceae. We compare the diversity of microbiota associated with the wild adult males of these two specialist fruit flies infesting different families of host plants. Targeted 16S rRNA gene (V3-V4 region) was sequenced using the Illumina MiSeq platform. Six bacterial phyla (Actinobacteria, Armatimonadetes, Bacteroidetes, Cyanobacteria/Melainabacteria group, Firmicutes, Proteobacteria) were detected at 97% similarity clustering and 0.001% abundance filtering. Four phyla (Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria) were present in all the specimens studied. Proteobacteria was the predominant phylum in both B. melastomatos and B. umbrosa. Enterobacteriaceae was the predominant family in UM B. melastomatos and B. umbrosa, and Orbaceae was the predominant family in Awana B. melastomatos. Klebsiella was the predominant genus in B. umbrosa, Citrobacter in UM B. melastomatos, and Orbus in Awana B. melastomatos. Double Wolbachia infections were present in UM B. melastomatos. In general, the bacterial diversity and richness varied within and between the samples of B. melastomatos and B. umbrosa.

Core members and differential abundance of chrysomelid microbiota in the life stages of Podontiaaffinis (Galerucinae) and adult Silanafarinosa(Cassidinae, Coleoptera).

The chrysomelid beetlesPodontiaaffinis and Silanafarinosa are members of the subfamilies Galerucinae and Cassidinae, respectively. This study, based on 16S rRNA gene-targeted metagenomics sequencing, reports the core members and differential abundance of bacterial communities in the larvae and adult beetles of P.affinis and the adult S.farinosa. Cyanobacteria/Melainabacteria group was the predominant phylum in the larvae of P.affinis, while Proteobacteria was the predominant phylum in adult P.affinis and S.farinosa. The number of Order, Family, Genus and Species OTUs in the adult stage of P.affinis was higher than that in the larval stage. The bacterial species richness of adult P.affinis was significantly higher than that of adult S.farinosa. Betaproteobacteria was the predominant class in adult P.affinis, Cyanobacteria in the larvae of P.affinis and Gammaproteobacteria in S.farinosa. The larvae and adult beetles of P.affinis and adult S.farinosahad a low number of unique and shared bacterial OTUs (> 5% relative abundance). The differences in the microbiota indicate possible differences in nutrient assimilation, host taxonomy and other stochastic processes. These findings provide new information to our understanding of the bacteria associated with specialist phytophagous chrysomelid beetles and beetles in general.

Complete mitochondrial genomes and phylogenetic relationships of the genera Nephila and Trichonephila (Araneae, Araneoidea).

Spiders of the genera Nephila and Trichonephila are large orb-weaving spiders. In view of the lack of study on the mitogenome of these genera, and the conflicting systematic status, we sequenced (by next generation sequencing) and annotated the complete mitogenomes of N. pilipes, T. antipodiana and T. vitiana (previously N. vitiana) to determine their features and phylogenetic relationship. Most of the tRNAs have aberrant clover-leaf secondary structure. Based on 13 protein-coding genes (PCGs) and 15 mitochondrial genes (13 PCGs and two rRNA genes), Nephila and Trichonephila form a clade distinctly separated from the other araneid subfamilies/genera. T. antipodiana forms a lineage with T. vitiana in the subclade containing also T. clavata, while N. pilipes forms a sister clade to Trichonephila. The taxon vitiana is therefore a member of the genus Trichonephila and not Nephila as currently recognized. Studies on the mitogenomes of other Nephila and Trichonephila species and related taxa are needed to provide a potentially more robust phylogeny and systematics.

Molecular differentiation and phylogenetic relationships of three Angiostrongylus species and Angiostrongylus cantonensis geographical isolates based on a 66-kDa protein gene of A. cantonensis (Nematoda: Angiostrongylidae).

The phylogenetic relationships and molecular differentiation of three species of angiostrongylid nematodes (Angiostrongylus cantonensis, Angiostrongylus costaricensis and Angiostrongylus malaysiensis) were studied using the AC primers for a 66-kDa protein gene of A. cantonensis. The AC primers successfully amplified the genomic DNA of these angiostrongylid nematodes. No amplification was detected for the DNA of Ascaris lumbricoides, Ascaris suum, Anisakis simplex, Gnathostoma spinigerum, Toxocara canis, and Trichinella spiralis. The maximum-parsimony (MP) consensus tree and the maximum-likelihood (ML) tree both showed that the Angiostrongylus taxa could be divided into two major clades - Clade 1 (A. costaricensis) and Clade 2 (A. cantonensis and A. malaysiensis) with a full support bootstrap value. A. costaricensis is the most distant taxon. A. cantonensis is a sister group to A. malaysiensis; these two taxa (species) are clearly separated. There is no clear distinction between the A. cantonensis samples from four different geographical localities (Thailand, China, Japan and Hawaii); only some of the samples are grouped ranging from no support or low support to moderate support of bootstrap values. The published nucleotide sequences of A. cantonensis adult-specific native 66kDa protein mRNA, clone L5-400 from Taiwan (U17585) appear to be very distant from the A. cantonensis samples from Thailand, China, Japan and Hawaii, with the uncorrected p-distance values ranging from 26.87% to 29.92%.

Estimating Human Exposure to Rat Lungworm (Angiostrongylus cantonensis) on Hawai'i Island: A Pilot Study.

Angiostrongylus cantonensis is a zoonotic, parasitic nematode causing angiostrongyliasis or rat lungworm disease. Clinical diagnosis in humans is currently confirmed by detection of parasite DNA in cerebrospinal fluid. This study estimated human exposure to A. cantonensis in volunteer participants solicitated via public venues on east Hawai'i Island using blood-based tests. Antibodies were screened in sera by crude antigen ELISA, followed by a 31-kDa dot-blot test developed and validated in Thailand. Human participants (n = 435) donated blood samples and completed a questionnaire to self-report relevant symptomology or clinical diagnosis. Among symptoms reported by participants diagnosed by licensed clinicians, headaches, high eosinophil counts, stiff neck, fatigue, and joint pain were most severe during the initial 3 months of infection. ELISA results revealed 22% of the serum samples as positive, 46% as equivocal, and 32% as negative. A subset of 186 samples was tested by dot blot, with 30% testing positive and 70% testing negative. A significantly higher mean ELISA value was found among recently (2014-2015) clinically diagnosed participants as than among those with a diagnosis before 2010 (P = 0.027). All dot-blot positives were also ELISA positive and were significantly associated with higher ELISA values compared with dot-blot negatives (P = 0.0001). These results suggest that an ELISA using crude antigen isolated from adult A. cantonensis from Hawai'i may be an effective initial screening method for estimating exposure to A. cantonensis in Hawai'i and likewise suggest that dot-blot tests using the 31-kDa antigen exhibit efficacy as a diagnostic for exposure.

Thai koi-hoi snail dish and angiostrongyliasis due to Angiostrongylus cantonensis: Effects of food flavoring and alcoholic drink on the third-stage larvae in infected snail meat.

Human infection with the rat lungworm Angiostrongylus cantonensis (Parastrongylus cantonensis) in Thailand, especially in the northeastern region, is associated with the habit of eating koi-hoi, which contains raw snail meat. Infection results from the snails being carriers of the larval parasite. The present study was conducted to assess the effect of food flavorings in koi-hoi, alcohol, and exposure time of the two variable on the infective larvae of A. cantonensis. Infected Biomphalaria glabrata snails were used for koi-hoi preparation. Raw snail meat was mixed with koi-hoi flavoring and left at room temperature for various time periods ranging from 5 to 60 minutes. At a predetermined time, two pieces of snail meat were removed at random and examined for viability (as determined by motility) of the parasitic third-stage larvae. At the same time, two random pieces of snail meat were removed and treated with 10 mL of a local 40% alcoholic drink for 30 minutes before examination of larval viability. Exposure of infected snail meat for 10 minutes or more to koi-hoi food flavoring resulted in significantly more nonmotile (dying or dead) larvae. Addition of the local alcoholic drink after exposure to the flavoring exerted an additional killing effect on the larvae. Despite long exposure time, both the koi-hoi flavoring and addition of alcoholic drink were not completely effective in killing the infective larvae in the snail meat. Thorough cooking of the food intended for human consumption should still be practiced.

Laboratory evaluation of aromatic essential oils from thirteen plant species as candidate repellents against Leptotrombidium chiggers (Acari: Trombiculidae), the vector of scrub typhus.

Scrub typhus, a rickettsial disease transmitted by several species of Leptotrombidium chiggers (larvae), is endemic in many areas of Asia. The disease is best prevented by the use of personal protective measures, including repellents. In this study commercially produced aromatic, essential oils of 13 plant species and ethanol (control) were tested in the laboratory for repellency against host-seeking chiggers of Leptotrombidium imphalum Vercammen-Grandjean and Langston (Acari: Trombiculidae). A rapid, simple and economic in vitro test method was used by exposing the chigger for up to 5 min. Repellency was based on relative percentages of chiggers attracted to test and control substances. Four of the 13 essential oils showed promise as effective repellent against L. imphalum chiggers. Syzygium aromaticum (clove) oil exhibited 100% repellency at 5% concentration (dilution with absolute ethanol), whereas Melaleuca alternifolia (tea tree) oil exhibited 100% repellency at 40% concentration. Undiluted oils of Zingiber cassamunar (plai) and Eucalyptus globules (blue gum) exhibited 100% repellency. Of the remaining nine essential oils, only 100% Pelargonium graveolens (geranium) exhibited >50% repellency (viz. 57%). Styrax torkinensis (benzoin) oil did not exhibit any repellency. These findings show that several aromatic, essential oils of plants may be useful as chigger repellent for the prevention of scrub typhus. Syzygium aromaticum oil may be safer and more economical to prevent chigger attacks than commercially available synthetic chemicals, such as DEET that may have harmful side effects.

Molecular phylogeography and genetic diversity of Angiostrongylus cantonensis and A. malaysiensis (Nematoda: Angiostrongylidae) based on 66-kDa protein gene.

Angiostrongylus cantonensis is the main causative agent of human angiostrongyliasis. A sibling species, A. malaysiensis has not been unequivocally incriminated to be involved in human infections. To date, there is only a single report on the application of the partial 66-kDa protein gene sequence for molecular differentiation and phylogeny of Angiostrongylus species. Nucleotide sequences of the 66-kDa protein gene of A. cantonensis and A. malaysiensis from Thailand, as well as those of the laboratory strains of A. cantonensis from Thailand and Hawaii, A. cantonensis from Japan and China, A. malaysiensis from Malaysia, and A. costaricensis from Costa Rica, were used for the reconstruction of phylogenetic tree by the maximum likelihood (ML) method and the haplotypes by the median joining (MJ) network. The ML phylogenetic tree contained two major clades with a full support bootstrap value - (1) A. cantonensis and A. malaysiensis, and (2) A. costaricensis. A. costaricensis was basal to A. cantonensis and A. malaysiensis. The genetic distance between A. cantonensis and A. malaysiensis ranged from p = .82% to p = 3.27%, that between A. cantonensis and A. costaricensis from p = 4.90% to p = 5.31%, and that between A. malaysiensis and A. costaricensis was p = 4.49% to p = 5.71%. Both A. cantonensis and A. malaysiensis possess high 66-kDa haplotype diversity. There was no clear separation of the conspecific taxa of A. cantonensis and A. malaysiensis from different geographical regions. A more intensive and extensive sampling with larger sample size may reveal greater haplotype diversity and a better resolved phylogeographical structure of A. cantonensis and A. malaysiensis.

Immunochromatographic test for rapid serological diagnosis of human angiostrongyliasis.

OBJECTIVES: The serological diagnosis of human infection with Angiostrongylus cantonensis remains problematic because there are no commercially available validated tests. Most laboratories use domestically prepared tests such as the enzyme-linked immunosorbent assay (ELISA) or immunoblotting. Since laboratory facilities are not always available in endemic areas, we developed and assessed a rapid lateral flow immunochromatographic assay (AcQuickDx Test) to detect anti-A. cantonensis antibodies in human serum. METHODS: The test device was assembled with purified 31-kDa glycoprotein as diagnostic antigen and with gold-labelled anti-human immunoglublin-G as the detector reagent. A total of 97 serum samples were tested - 19 samples from clinically diagnosed patients with detectable A. cantonensis-specific antibody in immunoblotting; 43 samples from patients with other parasitic diseases, i.e. gnathostomiasis (n=13), toxocariasis (n=2), trichinellosis (n=2), hookworm infection (n=4), filariasis (n=5), cysticercosis (n=9), paragonimiasis (n=2), opisthorchiasis (n=3), and malaria (n=3); and 35 samples from normal healthy subjects. RESULTS: The sensitivity, specificity, positive predictive value and negative predictive value of AcQuickDx Test to detect anti-A. cantonensis specific antibodies in serologically confirmed angiostrongyliasis cases, were 100%, 98.72%, 95% and 100%, respectively. Positive AcQuickDx was observed in 1 of 4 cases with hookworm infections. No positive AcQuickDx was observed in cases with other parasitic diseases, and the individual healthy subjects. CONCLUSIONS: AcQuickDx Test is rapid, highly sensitive and specific, and easy to perform without additional equipment or ancillary supplies. It yields results that are interpreted visually, and possesses a long shelf-life at room temperature. Thus, it can be applied as an additional test for clinical diagnostic support of angiostrongyliasis either in conventional laboratories or for remote areas where laboratory infrastructure is not available.