In vitro adhesion and invasion by Cladosporium sphaerospermum in human bronchial epithelial cells (BEAS-2B) and human pulmonary alveolar epithelial cells (HPAEpiC)

@#Cladosporium spores are ubiquitous in indoor and outdoor environment and may potentially trigger allergic responses upon inhalation. To date, there is limited investigation on the fate of Cladosporium spores after being inhaled into the respiratory tract. This study was conducted to investigate the interaction of Cladosporium sphaerospermum with Human Bronchial Epithelial Cells (BEAS-2B) and Human Pulmonary Alveolar Epithelial Cells (HPAEpiC). C. sphaerospermum conidia were harvested and co-cultured with BEAS-2B or HPAEpiC cells for 72 hours. At each time point (30 minutes, 2, 4, 24, 48 and 72 hours), adherence and invasion of the cells by C. sphaerospermum conidia (and hyphae) were investigated by immunofluorescence staining. This study demonstrated the adherence and internalization of C. sphaerospermum conidia within these epithelial cells. In addition, the conidia were able to germinate and invade the epithelial cells. The ability of the fungal conidia to adhere, internalize, germinate and invade both the bronchial and alveolar epithelial cells of the respiratory tract in vitro might contribute to the understanding of the pathogenesis of Cladosporium in respiratory infection and allergy in vivo. INTRODUCTION Cladosporium species is a member of the phylum Ascomycota. The common species include C. herbarum, C. cladosporioides and C. sphaerospermum. This genus has worldwide distribution. Aerobiological studies reported that majority of fungal spores in outdoor air is from the phyla Ascomycota and Basidiomycota, while Cladosporium is one of the most studied allergenic Ascomycetes fungi (Knutsen et al., 2012). Cladosporium spores are found abundantly in indoors and outdoors at approximately 18/m3 and 141/m3 respectively (Codina et al., 2008). As an imperfect dematiaceous fungus, Cladosporium species causes opportunistic infections such

Morphology of Glycycometus malaysiensis, a domestic mite in Malaysia

@#Glycycometus malaysiensis is an allergenic domestic mite found in houses. G. malaysiensis is known to be highly similar to and is often mistaken as Blomia tropicalis, one of the major house dust mite species that causes asthma and allergic diseases in many tropical and subtropical regions. It was also suggested that these mites cross-react with each other and that the prevalence of G. malaysiensis might be higher than previous reports. A review on the taxonomic keys as well as light and scanning electron micrographs of G. malaysiensis are presented to appreciate the fine morphological structures of G. malaysiensis. The mouth, setae, legs (trochanter, femur, genu, tibia and tarsus) and the sexual organs (genital openings, genital setae and genital suckers) of G. malaysiensis are outlined. The morphology of G. malaysiensis is also compared with that of B. tropicalis to delineate the key features for the differentiation between these two mite species.

Parasites in soil samples from Signy island, Antarctica

@#Studies on parasite populations in Antarctic soils are scarce and thus little is known about the threat of these parasites towards either the natural fauna or human visitors. However, human presence in Antarctica, mainly through research and tourism, keeps increasing over time, potentially exposing visitors to zoonotic infections from Antarctic wildlife and environment. Most available literature to date has focused on faecal samples from Antarctic vertebrates. Therefore, this study addressed the possible presence of parasites in Antarctic soil that may be infectious to humans. Soil samples were obtained from five locations on Signy Island (South Orkney Islands, maritime Antarctic), namely North Point and Gourlay Peninsula (penguin rookeries), Pumphouse (relic coal-powered pump house), Jane Col (barren high altitude fellfield) and Berntsen Point (low altitude vegetated fellfield close to current research station). Approximately 10% of the soil samples (14/135) from 3 out of the 5 study sites had parasites which included Diphyllobotridae spp. eggs, Cryptosporidium sp., an apicomplexan protozoa (gregarine), Toxoplasma gondii, helminths (a cestode, Tetrabothrius sp., and a nematode larva) and mites. The presence of parasites in the 3 sites are most likely due to the presence of animal and human activities as two of these sites are penguin rookeries (North Point and Gourlay Peninsula) while the third site (Pumphouse Lake) has human activity. While some of the parasite species found in the soil samples appear to be distinctive, there were also parasites such as Cryptosporidium and Toxoplasma gondii that have a global distribution and are potentially pathogenic.

Detection of dengue viruses and Wolbachia in Aedes aegypti and Aedes albopictus larvae from four urban localities in Kuala Lumpur, Malaysia

Dengue fever (DF) is currently one of the most important mosquito-borne diseases that affects humans. Dengue fever (DF) and dengue hemorrhagic fever (DHF) are caused by four serotypes of dengue viruses (DENV-1 to DENV-4). The main vector transmitting dengue is Aedes aegypti while Aedes albopictus acts as a secondary vector. As treatment is unavailable and the first dengue vaccine approved in Mexico, Dengvaxia® has yet to be accepted worldwide, prevention of the disease relies heavily on surveillance and control of mosquito vectors. A transgene driver, Wolbachia was found to limit the transmission of dengue virus in Aedes mosquitoes. Wolbachia alone was able to inhibit viral replication, dissemination and transmission in A. aeygpti mosquitoes in experimental studies. In A. albopictus, Wolbachia did not affect the replication of dengue virus but was able to reduce the viral infection of mosquito salivary glands and limit transmission. Studies on Wolbachia have all been carried out in adult Aedes mosquitoes, hence this study was conducted to determine the presence of dengue virus serotypes and Wolbachia in A. aegypti and A. albopictus larvae collected from ovitraps in four localities in Kuala Lumpur viz. Happy Gardens, IMU Bukit Jalil, Ampang and Taman Yarl. Another objective of this study was to determine the association between dengue virus serotypes and the presence of Wolbachia in A. aegypti and A. albopictus larvae. A total of 300 mosquito larvae was collected; 99 (Happy Gardens), 85 (Bukit Jalil), 73 (Ampang) and 43 (Taman Yarl). Out of 300 larvae collected, 284 were identified as A. albopictus and 16 others were identified as A. aegypti. Of the 284 A. albopictus larvae collected, 211 (74.3%) and 73 (25.7%) were found to be negative and positive for dengue virus respectively. The dengue serotypes detected were 2 DENV-2 (2.7%), 58 DENV-3 (79.5%) and 13 DENV-4 (17.8%). DENV-1 was not detected in any of the A. albopictus larvae. For A. aegypti, out of 16 A. aegypti larvae collected, 12 (75%) were found to be negative and 4 (25%) were positive for DENV-2. For the detection of Wolbachia in A. albopictus, 71 out of 284 (25%) and 213 (75%) larvae were found to be positive and negative for Wolbachia respectively. For A. aegypti, 4 (25%) and 12 (75%) out of 16 larvae were positive and negative for Wolbachia respectively. This is the first report of Wolbachia in A. albopictus and A. aegypti larvae in Malaysia. A chisquare test analysis to determine the association between dengue virus and Wolbachia in A. albopictus and A. aegypti larvae collected from the four localities in Kuala Lumpur showed that there was no association (χ2 = 3.080; df = 1; P > 0.05).

Sero-prevalence study of IgE responses to allergens from Malaysian house dust (HDM) and storage mites (SM)

Allergens of Dermatophagoides and Blomia species are well-characterized but not for other species. This study was conducted to determine the prevalence of allergic sensitization to house dust (HDM) and storage mites (SM). One hundred adult subjects (aged > 18) were recruited. The mite specific IgE of all allergic subjects were higher compared with healthy subjetcs despite being not statistically significant except for D. farinae and G. malaysiensis. The mean serum IgE levels against HDM and SM for allergic subjects were significantly higher compared with those in healthy subjects. They were mainly sensitized to Dermatophagoides farinae (35%) and Glycycometus malaysiensis (37%). Immunoblots revealed not all allergic subjects showed positive immuno-reactivity against the mites tested. Single or multiple bands were observed for different species. The subjects were commonly sensitized to Group 2 (9-12 kDa), 10 (38 kDa) and 18 (40-48 kDa) allergens. Twenty-one out of 60 allergic subjects were sensitized to either one or more species. The majority of them (71%) were sensitized to single species. The allergic subjects were mainly sensitized to D. pteronyssinus, followed by Tyrophagus putrecentiae and Aleuroglyphus ovatus. Seven were solely sensitized to HDM while 10 were solely sensitized to SM. Four subjects were sensitized to both. Preadsorption study revealed no cross-reactivity. There was difference between the prevalence and reactivity to allergens of HDM and SM in these subjects. Both ELISA and immunoblot did not correlate well but can complement each other in improving the detection of mite allergens to the species level.

Antigenic profile of Blomia tropicalis, Aleuroglyphus ovatus and Glycycometus malaysiensis

House dust mites and storage mites are well-known causes for allergenic diseases. The aim of this study was to investigate the immunogenic sites of Blomia tropicalis, Aleurogyphus ovatus and Glycycometus malaysiensis. The mites were maintained in a culture medium at 25ºC and 75% relative humidity. Mites were harvested either with heat escape or floatation method, purified, homogenized, quantified and used for the production of polyclonal antibody and immunostaining. For each species of mites, five male mice and five male rats were randomly selected and immunized intraperitoneally with respective crude mite extract at two-weekly intervals. Blomia tropicalis, A. ovatus or G. malaysiensis whole mites and paraffin-embedded mite sections were immunostained with the respective polyclonal antibody. The faecal pellets of mites were intensely stained for all the three species in the present study. The legs of sectioned A. ovatus were not immunogenic as compared with those of G. malaysiensis and B. tropicalis. The outer layer (cuticle) of whole mites and the eggs for these species were very immunogenic. Hence, the polyclonal antibodies obtained in this study may serve as potential tools in detecting the eggs and immature mites in environmental samples. Future studies should focus on the antigenic components of eggs since they were relatively abundant in dust and highly antigenic as seen in the present study.

Restriction enzyme digestion analysis of PCR-amplified DNA of Blastocystis hominis isolates.

Genomic DNA of Blastocystis isolates released into 0.1% Triton X-100 was suitable for amplification and yielded similar results as the genomic DNA extracted with standard kit. The specific B. hominis primers (BH1: GCT TAT CTG GTT GAT CCT GCC AGT and BH2: TGA TCC TTC CGC AGG TTC ACC TAC A) successfully produced the PCR product of about 1,770 bp with all the 7 Blastocystis isolates tested. The restriction fragment length polymorphism (RFLP) patterns yielded by 13 out of 25 restriction endonucleases showed that the 7 isolates could be grouped into 4 subgroups: subgroup-1 consisted of isolate C; subgroup-2 of isolates H4 and H7; subgroup-3 of isolates KP1, Y51 and M12; and subgroup-4 of isolate 27805. The differences between subgroups manifested as clear-cut RFLP patterns. A common band of 230 bp was revealed by Eco R1 in all the Blastocystis isolates tested. The band of about 180 bp was revealed by Alu I, differentiated symptomatic from asymptomatic isolates of this parasite, and might indicate the pathogenicity of this parasite.

New mechanical disruption method for extraction of whole cell protein from Candida albicans.

Cell disruption or lysis is a crucial step to obtain cellular components for various biological studies. We subjected different concentrations of Candida albicans to 5, 10, 15 and 20 cycles of disruption. The degree of cell lysis was observed using light microscopy and the yields obtained were measured and analysed. The optimum extraction with 1 x 10(10) yeast cells/ml was achieved after 5 cycles of disruption with 1.0 mm diameter glass beads at 5,000 rpm. Approximately 80% of the cells were lysed and the protein yield was 6,000 microg/ml. SDS-PAGE analysis revealed approximately 25 distinct protein bands with molecular weights ranging from 8 kDa to 220 kDa. We conclude that this mechanical disruption of fungal cells is a rapid, efficient and inexpensive technique for extracting whole cell proteins from yeast cells.

Polypeptides associated with in vitro cyst formation of Blastocystis hominis.

The objective of this study was to characterize the polypeptides associated with cysts of Blastocystis hominis. This form is believed to be infective and plays a role in parasite resistance to anti-B. hominis drugs currently used for treatment of Blastocystis associated diarrhea. Cysts were induced through in vitro culture of the parasite in complete medium supplemented with bacterial extract with trypticase, metronidazole or doxycycline. SDS-PAGE analysis showed almost similar polypeptide patterns of parasite extracts obtained from in vitro cultured parasites before and after exposure with the three supplements. Polypeptide bands at 76, 58.5, 48, 45, 40, 38, 32, 25 and 22 kDa were constantly seen in all antigenic preparations and no specific cyst-associated polypeptide was present. However, on immunoblot analysis, 3 out of 16 blastocystosis human sera identified a cyst-associated polypeptide at 60 kDa in all parasite extracts prepared from cultures with the three supplements. In addition, there were associated morphological changes detected in these parasites stained with acridine orange and observed under fluorescence microscopy. Metronidazole induced cyst forms (reddish cells) as early as 12 hours post-exposure; more cyst production (with stronger immunoblot bands) occurred after 24 hours exposure. However, cysts rupture with release and destruction of B. hominis daughters cells occurred after 48 hours exposure. Doxycycline induced less cyst-like forms at 24 hours (weaker 60 kDa band) and less destruction of the cysts (60 kDa band still present at 72 hours post exposure). Bacterial extract and trypticase also induced cysts at 12 hours with increasing numbers up to 72 hours exposure (corresponding increase in intensity of 60 kDa band from samples harvested at 12 to 72 hours post exposure) without any sign of deleterious effect on the parasite.

Detection of circulating antigens of Parastrongylus cantonensis in human sera by dot-blot ELISA and sandwich ELISA using monoclonal antibody.

A dot-blot ELISA was compared with a previously performed sandwich ELISA for the detection of Parastrongylus cantonensis antigens in sera from patients. Using the same monoclonal antibody and the same sera, 6 of 10 sera (60%) from parastronglyiasis patients were positive in dot-blot ELISA, whereas with sandwich ELISA, 5 of the same patient sera (50%) were positive. The specificity in both assays was 100% using 50 sera from patients with other parasitic diseases; of these, 10 each were from patients with cysticercosis, filariasis, gnathostomiasis, malaria and toxocariasis. The control group consisted of 53 sera from normal health Thais and Malaysians. The sensitivity of the assays was, however, slightly better with dot-blot ELISA and because it is simple, quick and cost-effective, it may be a test of choice for specific diagnosis of human parastrongyliasis.

Detection of Angiostrongylus malaysiensis circulating antigen using monoclonal antibody-based enzyme-linked immunosorbent assay (MAb-ELISA).

Three MAbs 1C4.2D8, 1C4.2C4 and 1C4.1F5 were produced using sonicated adult worm antigens of Angiostrongylus malaysiensis and they were found to be secreters of IgG1. The MAbs 1C4.2C4 and 1C4.2D8 were found to react with antigens of A. malaysiensis and cross-react with the closely related A. cantonensis but not with other helminths. A total of 108 human sera collected from Orang Asli (aborigenes) from Grik, in the State of Perak were tested for A. malaysiensis infection using the MAb-ELISA. MAb 1C4.1F5 and 25 (23%) were positive. Twenty of these positive samples were tested with the MAb 1C4.2D8 and none was found to be positive.

Detection of circulating antigens of Parastrongylus cantonensis in human sera by sandwich ELISA with specific monoclonal antibody.

A specific monoclonal antibody (AW-3C2) as revealed by ELISA was produced against the adult worm antigens of Parastrongylus cantonensis and used in a sandwich ELISA for the detection of circulating antigens in the sera of parastrongyliasis patients and those with other parasitic diseases. A total of 60 sera was used in this study. Of these, 10 each were from patients with parastrongyliasis filariasis, gnathostomiasis, malaria and toxocariasis. The control group consisted of 53 serum samples from normal healthy Thais and Malaysians. The mean +/- optical density (OD) values for the normal Thai and Malaysian groups were 0.126 +/- 0.028 and 0.124 +/- 0.029, respectively. Mean OD values of parastrongyliasis patient group differed significantly from that of the normal groups as well as those of other parasitic infections. Using a cut-off point of mean OD +/- 3SD of the normal control groups as indicating a positive reading, the specificity of the assay with this monoclonal antibody was 100% while the sensitivity was 50%.

Anopheles donaldi incriminated as a vector of periodic Brugia malayi in Grik, Perak, Malaysia.

Studies were carried out to observe the species composition of mosquitos and to determine the vectors responsible for the transmission of filariasis in Grik, Perak, Malaysia. A total of 2,155 mosquitos belonging to 7 genera and 30 species were collected. Anopheles donaldi comprised 24.1% of the collection. Twelve out of 519 An. donaldi were infected with L3 larvae of Brugia malayi. The peak biting time was around 23.00-24.00 hours. The infective bites per month ranged from 0 to 6.3.

In vitro drug susceptibility of Acanthamoeba castellani to chloroquine, ivermectin and fungizon.

In vitro sensitivity of Acanthamoeba castellani was tested to three drugs: Chloroquine, ivermectin and fungizone (amphotericin B). Sensitivity was demonstrated to the latter two compounds but not to chloroquine. Thus ivermectin and amphotericin B show promise as therapeutic agents against this parasite.

Seroprevalence of Sarcoptes scabiei var canis antibodies among aborigines in peninsular Malaysia.

The Aborigines or Orang Asli in Peninsular Malaysia who are still seminomadic are known to have a close association with dogs. In this study, enzyme-linked immunosorbent assay (ELISA) was used to detect anti-Sarcoptes scabiei var canis antibodies in this community as a measure of exposure to the mite. Out of 312 Orang Asli tested, 24.7% were positive for polyvalent anti-Sarcoptes antibodies. No significant difference was found between the positive rates in males (26.1%) and females (23.6%). Only 1.9% were positive for IgA and none was positive for IgE anti-Sarcoptes antibodies. Since there were very few patients with clinical manifestation of scabies, there is a possibility that continuous exposure to the dogs mite confers cross-protective immunity in the community against human scabies.

Detection of circulating antigens of Parastrongylus cantonensis in human sera by sandwich ELISA with specific monoclonal antibody.

A specific monoclonal antibody (AW-3C2) as revealed by ELISA was produced against the adult worm antigens of Parastrongylus cantonensis and used in a sandwich ELISA for the detection of circulating antigens in the sera of parastrongyliasis patients and those with other parasitic diseases. A total of 60 sera was used in this study. Of these, 10 each were from patients with parastrongyliasis, cysticercosis, filariasis, gnathostomiasis, malaria and toxocariasis. The control group consisted of 53 serum samples from normal healthy Thais and Malaysians. The mean +/- optical density (OD) values for the normal Thai and Malaysian groups were 0.126 +/- 0.028 and 0.124 +/- 0.029, respectively. The mean OD values of the parastrongyliasis patient group differed significantly from that of the normal groups as well as those of other parasitic infections. Using a cut-off point of OD +/- 3SD of the control groups as indicating a positive reading, the specificity of the assay with this monoclonal antibody was 100% while the sensitivity was 50%.

The impact of permethrin impregnated bednets on the malaria vector Anopheles maculatus (Diptera: Culicidae) in aboriginal villages of Pos Betau Pahang, Malaysia.

The effect of permethrin impregnated bednets on Anopheles maculatus Theobald was studied in four villages in Pos Betau, Pahang, Malaysia from August 1990 to July 1992. Collections of mosquitos were carried out indoors and outdoors from 1900 to 0700 hours. All mosquitos were dissected for sporozoites and parity. In May 1991 two villages received bednets impregnated with permethrin at 0.5 g/m2 and two villages received placebo bednets. There was a significant difference in the sporozoite and parous rates between the treated and control villages after the distribution of bednets (p < 0.05). There was no significant difference in the bites/man/night of An. maculatus between the pre and post treatment periods in the control villages. However there was a significant difference in bites/man/night between pre and post treatment in the treated villages (p < 0.001).