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Molecular identification of house dust mites and storage mites

Wong SF, Chong AL, Mak JW, Tan J, Ling SJ, Ho TM

Exp Appl Acarol, 2011 Oct;55(2):123-33.
PMID: 21468750 DOI: 10.1007/s10493-011-9460-6

Mites are known causes of allergic diseases. Currently, identification of mites based on morphology is difficult if only one mite is isolated from a (dust) sample, or when only one gender is found, or when the specimen is not intact especially with the loss of the legs. The purpose of this study was to use polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the ITS2 gene, to complement the morphological data for the identification of mites to the species level. For this, six species were cultured: Dermatophagoides pteronyssinus, D. farinae, Blomia tropicalis, Tyrophagus putrescentiae, Aleuroglyphus ovatus and Glycycometus malaysiensis. Genomic DNA of the mites was extracted, quantified, amplified and digested individually with restriction enzymes. Hinf I and Ple I differentiated the restriction patterns of D. pteronyssinus and D. farinae. Bfa I and Alu I enzymes differentiated B. tropicalis and G. malaysiensis. Ple I enzyme was useful for the differentiation between T. putrescentiae and A. ovatus. Bfa I was useful for the differentiation of G. malaysiensis from the rest of the species. In conclusion, different species of mites can be differentiated using PCR-RFLP of ITS2 region. With the established PCR-RFLP method in this study, identification of these mites to the species level is possible even if complete and intact adult specimens of both sexes are not available. As no study to date has reported PCR-RFLP method for the identification of domestic mites, the established method should be validated for the identification of other species of mites that were not included in this study.
Fulltext Serological screening of Tick-borne encephalitis (TBE) among Malaysian encephalitis patients

Thayan R, Khairullah NS, Ho TM

Trop Biomed, 2004 Dec;21(2):153-6.
PMID: 16493408

Tick-borne encephalitis (TBE) is a viral infection of the central nervous system and is caused by tick bites, usually after travel to rural or forested areas. The disease is prevalent in Scandinavia, Western Europe, Central Europe and the former Soviet Union and East Asia including Japan. In Malaysia, so far there are no reported cases of TBE. In the present time, many illnesses have been attributed to traveling to other parts of the world. Thus it is important to carry out TBE prevalence study to determine whether the virus is present among Malaysian population. Samples (sera and CSF) from patients admitted to major MOH hospitals in Peninsular Malaysia and Sabah with a clinical diagnosis of encephalitis but is IgM negative for JE, were tested for TBEV IgM ELISA and TBEV IgG ELISA (DRG, Germany). Out of the 600 samples screened for TBEV IgG, all were non-reactive. In addition, out of the 100 samples screened for TBEV IgM, all the samples were also non-reactive. Our results indicate that currently TBE is not present in the Malaysian population. Among the reasons for this could be lack of the infection agent, absence of the suitable vector or subjects selected for the study did not fit the criteria of possible exposure to TBE infections. Hence we recommend that for any future study, the selection of subjects should include those who returned from tick-infested forested areas.
Sequence analysis of the hypervariable regions of the 56 kDa immunodominant protein genes of Orientia tsutsugamushi strains in Malaysia

Tay ST, Rohani YM, Ho TM, Shamala D

Microbiol. Immunol., 2005;49(1):67-71.
PMID: 15665455

The DNA sequences encompassing two hypervariable regions, VD II and III of the 56 kDa immunodominant protein gene of 21 Malaysian strains of Orientia tsutsugamushi were determined. Two strains demonstrated a 100% DNA homology with the Gilliam prototype strain, and one with TH1817 strain and TA678 strain respectively. High percentages of DNA similarity (95-99%) were observed with Karp (4 strains), Gilliam (2 strains), TH1817 (4 strains), TC586 (3 strains) and TA763 (1 strain). The remaining strains demonstrated the highest DNA similarity with TA763 (1 strain, 89%), TA678 (1 strain, 86%) and TA686 (1 strain, 87%). Our study provides additional evidence on the existence and the genetic heterogeneity of TA strains of the Southeast Asia and their closely related strains in Malaysia.
Isolation and PCR detection of rickettsiae from clinical and rodent samples in Malaysia

Tay ST, Rohani MY, Ho TM, Devi S

Southeast Asian J. Trop. Med. Public Health, 2002 Dec;33(4):772-9.
PMID: 12757225

Isolation of rickettsiae from patients' blood samples and organ samples of wild rodents from areas with high seroprevalence of rickettsial infections was attempted using cell culture assay and animal passages. L929 mouse fibroblast cells grown in 24 well tissue culture plate were inoculated with buffy coat of febrile patients and examined for the growth of rickettsiae by Giemsa, Gimenez staining and direct immunofluorescence assay. No rickettsiae were isolated from 48 patients' blood samples. No symptomatic infections were noted in mice or guinea pigs infected with 50 organ samples of wild rodents. There was no rickettsial DNA amplified from these samples using various PCR detection systems for Orientia tsutsugamushi, typhus and spotted fever group rickettsiae.
Virulence of Malaysian isolates of Orientia tsutsugamushi in mice

Tay ST, Rohani MY, Ho TM, Devi S

Southeast Asian J. Trop. Med. Public Health, 2002 Sep;33(3):565-9.
PMID: 12693592

The pathogenicity of Malaysian isolates of Orientia tsutsugamushi was investigated by a mouse virulence assay. The isolates could be differentiated as low (4 isolates), moderately (3 isolates) and highly virulent (2 isolates) based on the different responses in infected mice. No direct correlation between severity of human scrub typhus infections and virulence of the O. tsutsugamushi in mice was observed. Mice infected with virulent strains of O. tsutsugamushi showed splenomegaly, ascitis accumulation and enlargement of kidneys and livers whereas avirulent O. tsutsugamushi strains were asymptomatic and exhibited ruffled fur for a short period after infection. There was low antibody response in mice infected with isolates of low pathogenicity as compared with those of highly virulent isolates. Upon dissection of the infected mice, enlargement of mouse organs such as spleen, kidney and liver was noted. Presence of rickettsemia in mice was confirmed by the growth of O. tsutsugamushi in the L929 cells when inoculated with blood from infected mice. O. tsutsugamushi was also cultured from the peritoneal exudates of the infected mice. However, DNA of O. tsutsugamushi was only detected in the peritoneal exudates (by PCR) and blood (by cell culture) and not from other tissue samples.
Antigenic types of Orientia tsutsugamushi in Malaysia

Tay ST, Rohani MY, Ho TM, Devi S

Southeast Asian J. Trop. Med. Public Health, 2002 Sep;33(3):557-64.
PMID: 12693591

The seroprevalence of various Orientia tsutsugamushi (OT) strains among Malaysian patients with suspected scrub typhus infections was determined using an indirect immunoperoxidase (IIP) assay. IgG against a single OT strain were detected in six sera (3 Karp, 1 Gilliam and 2 TC586), whereas IgM antibodies against a single OT strain (Gilliam) were noted in 3 sera (Gilliam). IgG reactive to all OT strains were present in 33 (47.1%) of the 70 sera and IgM reactive to all OT strains were present in 22 (78.6%) of the 28 sera. The fact that most sera were reactive to multiple OT strains suggests that group-specific antigens are involved in scrub typhus infections, whereas very few were due to strain-specific epitopes present on these strains. Peak IgG and IgM titers were noted more frequently against Gilliam, Karp, and TA763 strains: this suggests that these strains may be the commonest infecting strains among Malaysian patients. Two predominant OT polypeptides consistently reacted with patients' sera were the 70 kDa and 56 kDa proteins.
Expression of recombinant proteins of Orientia tsutsugamushi and their applications in the serodiagnosis of scrub typhus

Tay ST, Rohani MY, Ho TM, Devi S

Diagn Microbiol Infect Dis, 2002 Oct;44(2):137-42.
PMID: 12458119

In this study, recombinant proteins that encompassed the AD I-AD III regions of 56 kDa immunodominant gene of 2 Orientia tsutsugamushi (OT) serotypes; Gilliam and TA763 were expressed in Escherichia coli. Both recombinant proteins exhibited serologic cross-reactivity with the rabbit antisera against various OT serotypes, as evaluated by enzyme-linked immunosorbent assay (ELISA), but not against other rickettsial species, including Rickettsia typhi, R. prowazekii and TT118 SFG rickettsiae. The feasibility of using the recombinant proteins as a diagnostic reagent was further evaluated by ELISA using sera from blood donors and scrub typhus patients. The results suggested a higher affinity of the antihuman IgM than IgG with both recombinant proteins. The IgM ELISA findings were agreeable with the results of indirect immunoperoxidase (IIP) assay especially with sera of high antibody (1:1600). However, more than one antigen are probably needed for development of an effective assay for serodiagnosis of scrub typhus in endemic areas.
Antibodies to Orientia tsutsugamushi, Rickettsia typhi and spotted fever group rickettsiae among febrile patients in rural areas of Malaysia

Tay ST, Ho TM, Rohani MY, Devi S

Trans R Soc Trop Med Hyg, 2000 9 7;94(3):280-4.
PMID: 10974999

A serosurvey was conducted in 1995-97 among 1596 febrile patients from 8 health centres in Malaysia for antibodies against Orientia tsutsugamushi (OT), Rickettsia typhi (RT) and TT118 spotted fever group rickettsiae (SFGR) by using an indirect immunoperoxidase assay. A total of 51.4% patients had antibody against at least 1 of those rickettsiae. Antibody to SFGR was most prevalent (42.5%), followed by RT (28.1%) and OT (24.9%). The seroprevalences of antibodies to SFGR, RT or OT alone were 12.4, 3.6 and 4.3%, respectively. Antibodies against more than 1 species of rickettsiae were presence in 31.1% of the patients, suggesting the possibility of co-infection, previous exposures or serological cross-reactivities. Seroprevalence of the various rickettsiae varied according to locality, with SFGR antibodies being the most prevalent in most areas. There was no significant association of prevalence of rickettsial antibody with gender. The seroprevalence of OT, SFGR and RT increased with patient age but an increase of antibody titre with age was not significant. Those working in the agricultural sectors had significantly higher seroprevalence of OT, SFGR and RT than those not related with agricultural activities. Scrub typhus remains a public health problem with an estimated annual attack rate of 18.5%. Tick typhus and murine typhus as shown in this serosurvey appear much more widespread than scrub typhus in this country.
In vitro demonstration of the hemolytic, cytotoxic activities and induction of apoptosis in Orientia tsutsugamushi infected L929 mouse fibroblast cells

Tay ST, Rohani MY, Ho TM, Devi S

Southeast Asian J. Trop. Med. Public Health, 2003 Jun;34(2):352-6.
PMID: 12971561

Using cultured mouse fibroblast L929 cells, this study demonstrated the hemolytic and cytotoxic activities and induction of apoptosis in cells infected with Orientia tsutsugamushi. Low levels of hemolytic activity were detected using heavily infected cells. No hemolysin or cytotoxin were detected in the infected culture fluid regardless of the pathogenicity of the O. tsutsugamushi strains in mice. Using propidium iodide uptake assay, acridine orange/ethidium bromide staining and terminal deoxynucleotide transferase-mediated dUTP-digoxigenin nick-end labeling assay, apoptosis was observed in L929 cells infected with Karp and Gilliam strains.
Fulltext Serological evidence of natural infection of wild rodents (Rattus spp and Tupaia glis) with rickettsiae in Malaysia

Tay ST, Kaewanee S, Ho TM, Rohani MY, Devi S

Southeast Asian J. Trop. Med. Public Health, 1998 Sep;29(3):560-2.
PMID: 10437957
Fulltext Serological findings of Coxiella burnetii infection among patients with fevers in a health centre in Sarawak, Malaysia

Tay ST, Ho TM, Rohani MY

Southeast Asian J. Trop. Med. Public Health, 1998 Mar;29(1):94-5.
PMID: 9740277
Fulltext Use of hemolymph test for detection of rickettsiae in Malaysian ticks

Tay ST, Ho TM, Rohani MY

Southeast Asian J. Trop. Med. Public Health, 1996 Sep;27(3):642-4.
PMID: 9185285
Fulltext Antigenic profile of Blomia tropicalis, Aleuroglyphus ovatus and Glycycometus malaysiensis

Tang JC, Wong SF, Mak JW, Ho TM

Trop Biomed, 2011 Aug;28(2):223-36.
PMID: 22041741

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.
Residual activity of benzyl benzoate against Dermatophagoides pteronyssinus (Acari: Pyroglyphidae)

Suhaili ZA, Ho TM

Southeast Asian J. Trop. Med. Public Health, 2008 May;39(3):507-10.
PMID: 18564691

The residual effect of a commercial spray containing 2.0% benzyl benzoate against Dermatophagoides pteronyssinus was evaluated in the laboratory. Filter papers treated with benzyl benzoate were dried for 2 hours and placed in Petri dishes. Adult mites were then added and mortality was assessed following 24 hour contact and at succeeding 4 weeks intervals up to week 28 post-treatment using the same treated filter papers. There was 100% mortality at 24 hours post-treatment, declining progressively to 71% by week 28. Probit analysis estimated a median lethal time (LT50) of 59 weeks.
Fulltext Acariasis on pet Burmese python, Python molurus bivittatus in Malaysia

Mariana A, Vellayan S, Halimaton I, Ho TM

Asian Pac J Trop Med, 2011 Mar;4(3):227-8.
PMID: 21771459 DOI: 10.1016/S1995-7645(11)60075-8

OBJECTIVE: To identify the acari present on pet Burmese pythons in Malaysia and to determine whether there is any potential public health risk related to handling of the snakes.
METHODS: Two sub-adult Burmese pythons kept as pets for a period of about 6 to 7 months by different owners, were brought to an exotic animal practice for treatment. On a complete medical examination, some ticks and mites (acari) were detected beneath the dorsal and ventral scales along body length of the snakes. Ticks were directly identified and mites were mounted prior to identification.
RESULTS: A total of 12 ticks represented by 3 males, 2 females and 7 nymphal stages of Rhipicephalus sanguineus (R. sanguineus) were extracted from the first python while the other one was with 25 female Ophionyssus natricis (O. natricis) mesostigmatid mites. Only adult female mites were found. These mites are common ectoparasites of Burmese pythons.
CONCLUSIONS: Both the acarine species found on the Burmese pythons are known vectors of pathogens. This is the first record that R. sanguineus has been reported from a pet Burmese python in Malaysia.
Fulltext Acarine ectoparasites of Panti Forest Reserve in Johore, Malaysia

Mariana A, Mohd KB, Halimaton I, Suhaili ZA, Shahrul-Anuar MS, Nor ZM, et al.

Asian Pac J Trop Biomed, 2011 Jan;1(1):1-5.
PMID: 23569714 DOI: 10.1016/S2221-1691(11)60057-9

To identify the presence of acarine ectoparasites and determine whether there is any potential public health risk in Panti Forest Reserve, Johore, Malaysia.
Ticks (Ixodidae) and other ectoparasites in Ulu Muda Forest Reserve, Kedah, Malaysia

Mariana A, Zuraidawati Z, Ho TM, Kulaimi BM, Saleh I, Shukor MN, et al.

Southeast Asian J. Trop. Med. Public Health, 2008 May;39(3):496-506.
PMID: 18564690

A survey of ticks and other ectoparasites was carried out during a national biodiversity scientific expedition at Ulu Muda Forest Reserve, Kedah, Malaysia from 23-29 March 2003. A total of 161 animals comprising 20 species of birds, 16 species of bats, six species of non-volant small mammals and 12 species of reptiles were examined for ticks and other ectoparasites. From these animals, nine species in five genera of ticks, 10 species in two families of Mesostigmatid mites and five species of chiggers were collected. Three of the ectoparasitic species found, Dermacentor auratus, Ixodes granulatus and Leptotrombidium deliense are of known public health importance. This survey produced the first list of ticks and other ectoparasites in the forest reserve and the third study of ectoparasites in Kedah. Fourteen species of these ectoparasites are new locality records.
A survey of ectoparasites in Gunung Stong Forest Reserve, Kelantan, Malaysia

Mariana A, Zuraidawati Z, Ho TM, Mohd Kulaimi B, Saleh I, Shukor MN, et al.

Southeast Asian J. Trop. Med. Public Health, 2005 Sep;36(5):1125-31.
PMID: 16438136

A survey of ticks and other ectoparasites was carried out during a national biodiversity scientific expedition at Gunung Stong Forest Reserve, Kelantan, Malaysia from 23-29 May 2003. A total of 272 animals comprised of 12 species of birds, 21 species of bats, 7 species of rodents and 2 species of insects were examined for ticks and other ectoparasites. From these animals, 5 species in 4 genera of ticks; 7 species in 2 families of Mesostigmatid mites and 5 species of chiggers were collected. Among the ectoparasites found were Ixodes granulatus and Leptotrombidium deliense, which are of known medical importance. A tick island consisting of 10 nymphal stages of Dermacentor spp was observed feeding on Rattus tiomanicus.
First report on sensitization to allergens of a house dust mite, Suidasia pontifica (Acari: Saproglyphidae)

Mariana A, Ho TM, Gendeh BS, Iskandar H, Zainuldin-Taib M

Southeast Asian J. Trop. Med. Public Health, 2000 Dec;31(4):722-3.
PMID: 11414419

A species of house dust mite, Suidasia pontifica, was recently shown to produce allergens affecting man. The species may be as important as other allergen producing mite in sensitization and causing allergic symptoms in Malaysians. Surveys conducted demonstrated that 80% of the houses surveyed were positive for this mite with densities ranged from 2 to 50 mites per gram of dust. Colonies of the species has been successfully established and materials from those colonies have been used to produce extracts for studies on sensitization to the mites. A total of 85 suspected allergic rhinitis patients were tested and 74.1% demonstrated positive reactions. Extract of this mite should be considered for routine diagnostic testing and possible immunotherapy.
Fulltext House dust mite fauna in the Klang Valley, Malaysia

Mariana A, Ho TM, Sofian-Azirun M, Wong AL

Southeast Asian J. Trop. Med. Public Health, 2000 Dec;31(4):712-21.
PMID: 11414418

Allergy to house dust mites (HDM) is an important cause of asthma and rhinitis in Malaysia. This study was carried out to evaluate the dust mite fauna in the Klang Valley. Dust samples were collected from 20 houses from March 1994 to February 1995. Thirty-three dust samples from mattresses were examined monthly for the occurrence of HDM. A total of 22 species in 9 families of HDM was identified. The most common and densely populated species was Blomia tropicalis with an average density of 8,934 mites/g of dust. Dermatophagoides pteronyssinus was the next in abundance, followed by Malayoglyphus intermedius. All houses surveyed were found to be infested with HDM and every house had at least 6 species of HDM. B. tropicalis and D. pteronyssinus were found in all mattresses. HDM in the Klang Valley were found to be highly prevalent and present in high densities. In this study, counts of D. pteronyssinus was found to exceed the proposed exposure threshold of 500 mites/g dust, for triggering acute asthma. Although counts of B. tropicalis exceeded D. pteronyssinus, no conclusion could be made because there is currently no exposure threshold for triggering acute asthma, for this species. Monthly distribution of B. tropicalis and D. pteronyssinus showed 2 peaks and 4 peaks, respectively. The major peak for D. pteronysinus was in January 1995 whereas for B. tropicalis, the major peak was more variable and occurred between November 1994 to January 1995. Both the species showed minor peak in April 1994.

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