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Specificity and sensitivity of a rapid dipstick test (Brugia Rapid) in the detection of Brugia malayi infection

Rahmah N, Taniawati S, Shenoy RK, Lim BH, Kumaraswami V, Anuar AK, et al.

Trans R Soc Trop Med Hyg, 2002 1 31;95(6):601-4.
PMID: 11816429

A total of 753 serum samples from 6 institutions in 3 countries (Malaysia, Indonesia and India) were used to evaluate an immunochromatographic rapid dipstick test, Brugia Rapid, for diagnosis of Brugia malayi infection. The samples comprised sera from 207 microfilaria-positive individuals and 546 individuals from filaria non-endemic areas. The latter consisted of 70 individuals with soil-transmitted helminth infections, 68 with other helminth infections, 238 with protozoan infections, 12 with bacterial and viral infections and 158 healthy individuals. The dipstick is prepared with a goat anti-mouse antibody control line and a B. malayi recombinant-antigen test line. First, the dipstick is dipped into a well containing diluted patient serum, thus allowing specific anti-filarial antibody in the serum to react with the recombinant antigen. Then the dipstick is placed into an adjacent well containing reconstituted anti-human IgG4-gold. After 10 min, development of 2 red-purplish lines denotes a positive result and one line indicates a negative reaction. The overall results of the evaluation showed 97% sensitivity, 99% specificity, 97% positive predictive value and 99% negative predictive value. Brugia Rapid is thus a promising diagnostic tool for detection of B. malayi infection, and would be especially useful for the brugian filariasis elimination programme.
Fulltext Comparison of Brugia-Elisa and thick blood smear examination in a prevalence study of brugian filariasis in Setiu, Terengganu, Malaysia

Lim BH, Rahmah N, Afifi SAB, Ramli A, Mehdi R

Med J Malaysia, 2001 Dec;56(4):491-6.
PMID: 12014770

A total of 1,134 finger-pricked blood samples were collected from residents of Setiu, Terengganu. A drop of blood was used to make thick blood smear and about four drops were used for obtaining serum. The smears were stained and examined by the State Vector Control Unit in Kuala Terengganu, while the serum samples were tested for specific IgG4 antibodies to a novel recombinant antigen using Brugia-Elisa. Prevalence of filariasis in these areas were found to be 0.26% (3/1,134) using thick blood smear examination and 2.47% (28/1,134) using Brugia-Elisa, thus demonstrating the greater sensitivity of the latter test. In addtion, Brugia-Elisa showed a high level of specificity (97.8%, 1,106/1,131) when compared to thick blood smear examination.
A recombinant antigen-based IgG4 ELISA for the specific and sensitive detection of Brugia malayi infection

Rahmah N, Lim BH, Khairul Anuar A, Shenoy RK, Kumaraswami V, Lokman Hakim S, et al.

Trans R Soc Trop Med Hyg, 2001 8 9;95(3):280-4.
PMID: 11490997

An IgG4 ELISA based on a novel recombinant antigen was evaluated for detection of Brugia malayi infection, using 2487 sera from various institutions: 2031 samples from Universiti Sains Malaysia, 276 blinded sera from 2 other institutions in Malaysia, 140 blinded sera from India and 40 blinded sera from Thailand. These sera were from various groups of individuals, i.e., microfilaraemics, chronic patients, endemic normals, non-endemic normals and individuals with other parasitic and bacterial infections. Based on a cut-off optical density reading of 0.300, the IgG4 ELISA demonstrated specificity rates of 95.6-100%, sensitivity rates of 96-100%, positive predictive values of 75-100% and negative predictive values of 98.9-100%. These evaluation studies demonstrated the high specificity and sensitivity of this test for the detection of active B. malayi infection. Thus, the IgG4 ELISA would be very useful as a tool in diagnosis and in elimination programmes for brugian filariasis.
Fulltext Epidemiological screening of lymphatic filariasis among immigrants using dipstick colloidal dye immunoassay

Wan Omar A, Sulaiman O, Yusof S, Ismail G, Fatmah MS, Rahmah N, et al.

Malays J Med Sci, 2001 Jul;8(2):19-24.
PMID: 22893756

We have recently reported that a dipstick colloidal dye immunoassay (DIA) that detect parasite antigens in human serum is sensitive and specific for the diagnosis of active infection of lymphatic filariasis. Rabbit polyclonal antibodies (RbBmCAg) labelled with a commercial dye, palanil navy blue was used to detect filarial antigenemia among Indonesian and Bangladeshi immigrant workers (N= 630) at oil palm estates at Hulu Trengganu District, Peninsular Malaysia. Microfilaremia with Brugia malayi were detected in 51 (8.10 %) individuals, of which 42 (6.67 %) were among the Indonesians and 9 (1.98 %) among the Bangladeshis. Microfilaremia with Wuchereria bancrofti were detected in 33 (5.24 %) individuals of which 15 (2.38 %) were among the Indonesians and 18 (2.86 %) among the Bangladeshis workers. The DIA detected 96 (15.24 %) antigenemic cases which comprise of all the microfilaremic cases and 15 (2.38 %) amicrofilaremic cases. The amicrofilaremic cases with filarial antigenemia consisted of 9 (1. 43 %) Indonesians and 6 (0.95%) Bangladeshis. We have used 6 ul of the RbBmCAg and diluted (1:10) patients' sera per dipstick which make the DIA reagent conservative. The DIA is a rapid test and can be read in approximate 2 hours.. Additionally, coloured dots developed in the DIA can be qualitatively assessed visually for intensity. The DIA does not require sophisticated equipment or radioactivity, and therefore suitable for field application.
PCR-ELISA for the detection of Brugia malayi infection using finger-prick blood

Rahmah N, Ashikin AN, Anuar AK, Ariff RH, Abdullah B, Chan GT, et al.

Trans R Soc Trop Med Hyg, 1998 12 16;92(4):404-6.
PMID: 9850392

A polymerase chain reaction assay based on the enzyme-linked immunosorbent assay (PCR-ELISA) has been developed to detect Brugia malayi infection in an area of low endemicity in Malaysia. Blood samples from 239 subjects were tested: 192 amicrofilaraemic individuals, 14 microfilaraemic persons and 3 chronic elephantiasis cases from endemic areas and 30 city-dwellers (non-endemic controls). PCR products were examined by ELISA and Southern hybridization. In the PCR-ELISA, digoxigenin-labelled PCR products were hybridized to a biotin-labelled probe. This was followed by incubation in streptavidin-coated microtitre wells and detection using anti-digoxigenin-peroxidase and ABTS [2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonic acid)]. All microfilaraemic samples were positive by PCR-ELISA and Southern hybridization and all samples from non-endemic subjects and chronic elephantiasis patients were negative. The PCR-ELISA detected 12 times as many B. malayi infections as did thick blood film examination. Nineteen of the 194 samples from the endemic area gave positive results by both PCR-ELISA and Southern hybridization, and an additional 5 samples were positive by PCR-ELISA only. The PCR-ELISA was specific and sensitive, detected more infections, and was more reproducible than Southern hybridization.
A Brugia malayi antigen specifically recognized by infected individuals

Rahmah N, Anuar AK, A'shikin AN, Lim BH, Mehdi R, Abdullah B, et al.

Biochem Biophys Res Commun, 1998 Sep 29;250(3):586-8.
PMID: 9784388

Western blot analyses were performed on 444 serum specimens: 40 sera from microfilaraemic individuals, 10 sera from elephantiasis patients, 24 treated individuals, 50 sera from residents of endemic areas without anti-filarial IgG4 antibodies (endemic normals), 20 sera from amicrofilaraemic individuals with high anti-filarial IgG4 antibodies, 200 sera from healthy city-dwellers (non-endemic samples), and 100 sera from soil-transmitted helminth-infected individuals. Phast electrophoresis system was used to electrophorese Brugia malayi soluble adult worm antigen on 10-15% SDS-PAGE gradient gels followed by electrophoretic transfer onto PVDF membranes. Membrane strips were then successively incubated with blocking solution, human sera, and monoclonal anti-human IgG4 antibody-HRP, with adequate washings done in between each incubation step. Luminol chemiluminescence detection was then used to develop the blots. An antigenic band with the MW of approximately 37 kDa was found to be consistently present in the Western blots of all microfilaraemic sera, all amicrofilaraemic sera with high titres of anti-filarial IgG4 antibodies, some treated patients, and some elephantiasis patients. The antigen did not occur in immunoblots of individuals with other helminthic infections, normal endemic individuals, and city dwellers. Therefore the B. malayi antigen of with the MW of approximately 37 kDa demonstrated specific reactions with sera of B. malayi-infected individuals and thus may be useful for diagnostic application.
Use of antifilarial IgG4-ELISA to detect Brugia malayi infection in an endemic area of Malaysia

Rahmah N, Anuar AK, Ariff RH, Zurainee MN, A'shikin AN, Fadzillah A, et al.

Trop Med Int Health, 1998 Mar;3(3):184-8.
PMID: 9593356

OBJECTIVE: To evaluate the usefulness of antifilarial IgG4 antibody assay in detecting B. malayi infection in a filaria endemic area in Malaysia.
METHODS: A sandwich ELISA using B. malayi soluble antigen was employed to detect antifilarial IgG4 antibodies in serum samples of 330 individuals who comprised 88 healthy individuals from nonendemic areas, 15 B. malayi microfilaraemic cases, 22 individuals with soil-transmitted helminthiases, 9 elephantiasis cases and 196 residents from a B. malayi-endemic area. An O.D. value of > 0.420 at serum dilution of 1:400 was used as the cut-off point. This cut-off point was obtained by taking the mean optical density (0.252 + 4 S.E.) of 36 negative sera which had O.D. values greater than 0.1 at serum dilution of 1:400.
RESULTS: All 15 microfilaraemic persons were positive for antifilarial IgG4 antibody. Non-endemic normals, soil-transmitted helminth infected persons and chronic elephantiasis cases were negative for antifilarial IgG4 antibody. Of the 196 individuals from the filaria endemic area, 37 (18.8%) demonstrated presence of antifilarial IgG4 antibodies; and only eight individuals (4.1%) were positive for microfilariae. All eight microfilaraemic individuals were also positive for antifilarial IgG4 antibodies.
CONCLUSION: Antifilarial IgG4-ELISA could detect 4.6 times more positive cases than the microfilaria detection method. With appropriate cut-off values that eliminate cross-reactivities, this serological tool is very useful for Brugia malayi prevalence surveys and diagnosis.
Fulltext Parasitic infections among aborigine children at Post Brooke, Kelantan, Malaysia

Rahmah N, Ariff RHT, Abdullah B, Shariman MSM, Nazli MZM, Rizal MZM

Med J Malaysia, 1997 Dec;52(4):412-5.
PMID: 10968120

This study investigated the prevalence of parasitic infections among aborigine children at Post Brooke, Kelantan. Eighty-four formalin-fixed specimens and 78 PVA-fixed specimens were obtained. 79.8% and 35.9% of the samples were positive for helminth ova and protozoa respectively. The parasites detected (single plus mixed infections) were A. lumbricoides (50/84, 59.5%), T. trichiura (35/84, 41.7%), hookworm (5/84, 6.0%), S stercoralis (1/84, 1.2%), G. intestinalis (18/78, 23.1%), E. histolytica (7/78, 9.0%) and E. coli (7/78, 9.0%). Two hundred thick blood film examinations detected only one case of Plasmodium falciparum infection. A high prevalence of intestinal parasitic infections among the children at Post Brooke was demonstrated in this study; thus there is an urgent need to improve the hygiene, education and living standards of this population.
Potential use of IgG2-ELISA in the diagnosis of chronic elephantiasis and IgG4-ELISA in the follow-up of microfilaraemic patients infected with Brugia malayi

Rahmah N, Anuar AK, Karim R, Mehdi R, Sinniah B, Omar AW

Biochem Biophys Res Commun, 1994 Nov 30;205(1):202-7.
PMID: 7999024

Sera from fifty subjects with different presentations of Brugian filariasis and from common soil-transmitted helminth infections were tested for specific anti-filarial IgG and its subclasses. Anti-filarial IgG, IgG1 and IgG3 showed cross-reactivities with soil-transmitted helminthic infections and no significant differences in optical densities among the various groups of filarial patients. In comparison with other groups of subjects, IgG4-ELISA of sera from microfilaraemic patients and some previously microfilaraemic patients showed a significant increase in optical density readings, while IgG2-ELISA showed elevated optical density readings in sera of patients with chronic elephantiasis. Therefore IgG2-ELISA is potentially useful in the diagnosis of brugian chronic elephantiasis while IgG4-ELISA may be beneficial for follow-up diagnosis of treated microfilaraemic patients.
Comparison of three forms of antigens in the demonstration of cell-mediated immune response in murine toxoplasmosis

Rahmah N, Khairul Anuar A

Biochem Biophys Res Commun, 1992 Dec 15;189(2):640-4.
PMID: 1472034

Mice were chronically infected with cysts of ME49 strain of Toxoplasma gondii. At different periods post-infection, their spleens were removed and single cell suspensions were made. Lymphocyte transformation experiments were performed on the lymphocyte suspensions using three different kinds of antigens of ME49 strain of T. gondii, namely soluble, excretory/secretory and cystic forms. The results showed that the pattern of lymphocyte responsiveness was dependent on the kind of antigen employed for induction of the blastogenesis. Using soluble and cystic forms of the antigen, different periods of lymphocyte suppression and lymphocyte proliferation were demonstrated. However, with the use of excretory/secretory antigen, no significant suppression of lymphocyte stimulation was noted throughout the course of infection. Thus excretory/secretory antigen may be the best form of antigen for stimulation of the cell-mediated immune response and hence it appears to be a good candidate for vaccine in toxoplasmosis.
Demonstration of antigenic similarities and variations in excretory/secretory antigens of Toxoplasma gondii

Rahmah N, Anuar AK

Biochem Biophys Res Commun, 1992 Aug 31;187(1):294-8.
PMID: 1520310

C57BL/6 mice were orally infected with different doses of cysts of ME49 strain of Toxoplasma gondii to produce groups of acutely and chronically infected mice. Sera were obtained at different periods post-infection. SDS-PAGE was ran with excretory/secretory antigens of ME49 and RH strains of T. gondii, followed by Western blot analyses using the above sera and anti- IgA, IgM, IgG as conjugates. The SDS-PAGE profiles of the two antigens were similar. However the antigenic bands showed variations in all blots, most evidently in IgA blots of chronic sera. IgG blots showed greatest similarities in reactive bands. In IgM blots, more common bands were shown in chronic sera than in acute sera. Variations and similarities in prominence of some bands and time of their appearance were also noted, especially in IgM and IgG blots of chronic sera. Thus antigenic variations and similarities are present in excretory/secretory products of different strains of T. gondii.