A rapid antibody detection test is very useful for the detection of lymphatic filariasis, especially for certification and surveillance of post-mass drug administration. panLF Rapid kit is suitable for this purpose since it can detect all species of lymphatic filaria. It is based on the detection of anti-filarial IgG4 antibodies that react with recombinant B. malayi antigens, BmR1 and BmSXP. There is an increase demand for the test due to its attributes of being rapid, sensitive and specific results, as well as its field-applicability. The main aim of this paper is to obtain high recovery and purity of recombinant antigen BmSXP via a modified method of immobilized metal affinity chromatography (IMAC). The highest product yield of 11.82 mg/g dry cell weight (DCW) was obtained when IMAC was performed using the optimized protocol of 10 mM imidazole concentration in lysis buffer, 30 mM imidazole concentration in wash buffer, and 10 column volume wash buffer containing 300 mM salt concentration. This gave a 54% protein recovery improvement over the manufacturer's protocol which recorded a product yield of only 7.68 mg/g DCW. The recovered BmSXP recombinant antigen showed good western blot reactivity, high sensitivity (31/32, 97%) and specificity (32/32, 100%) in ELISA, thus attesting to its good purity and quality.
Traditionally serum and/or CSF specimens have been used for detection of either specific antibodies or antigens as a supportive diagnosis of NCC. However, in recent days, much interest has been shown employing noninvasive specimens such as urine. In our study, we identified and compared a profile of circulating antigenic peptides of parasite origin in three different body fluids (CSF, serum and urine) obtained from confirmed NCC cases and control subjects. The circulating antigenic peptides were resolved by SDS-PAGE and subjected to immunoblotting. For confirmation of their origin as parasite somatic or excretory secretory (ES) material, immunoreactivity was tested employing affinity purified polyclonal Taenia solium metacestode anti-somatic or ES antibodies, respectively. Only lower molecular weight antigenic peptides were found circulating in urine in contrast to serum and CSF specimens. Few somatic peptides were identified to be 100% specific for NCC (19·5 kDa in all three specimens; 131, 70 kDa in CSF and serum only; 128 kDa in CSF only). Similarly, the specific ES peptides detected were 32 kDa (in all three specimens), 16·5 kDa (in serum and CSF only), and 15 kDa (urine only). A test format detecting either one or more of these specific peptides would enhance the sensitivity in diagnosis of NCC.
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.
Wuchereria bancrofti, Brugia malayi and Brugia timori are the causative agents of lymphatic filariasis in Indonesia but in some endemic areas, B malayi is more commonly found. Diagnosis of filariasis is normally based on clinical, parasitological and immunological examinations but those methods have limitations. The discovery of monoclonal antibodies is expected to provide a new dimension to the efforts in the development of specific and sensitive immunological tests for the various stages of filariasis infection. This preliminary report, using monoclonal antibodies and dot-blot assay in human lymphatic filariasis showed that 75% of sera from microfilaremic patients with clinical signs, 40% of sera from amicrofilaraemic patients with clinical signs, 88.8% of sera from microfilaremic patients without clinical signs and 19.6% of sera from amicrofilaremic patients without clinical signs have circulating antigens.
A multicentre evaluation of the Brugia Rapid dipstick test was performed using 1263 serum samples in four international laboratories, i.e. T.D. Medical College (TDMC, India), National Institutes of Health (NIH, USA), Swiss Tropical Institute (STI, Switzerland) and Leiden University Medical Centre (LUMC, Netherlands). In comparison with microscopy, the dipstick demonstrated sensitivities of 97.2% (70 of 72) at TDMC, 91.6% (175 of 191) at LUMC and 100% (six of six) at STI. Sera of chronic patients showed a positivity rate of 11.3% (19 of 168) and 61.2% (71of 116) at TDMC and LUMC, respectively. All 266 sera of non-endemic normals from STI, NIH and LUMC tested negative with the dipstick. At LUMC, sera of 'endemic normals' (amicrofilaraemics with no clinical disease) from an area with approximately 35% microfilaria positivity showed 60.8% positive results (31 of 51), thus demonstrating the likelihood of many cryptic infections occurring in this population. Specificities of the test with Onchocerca volvulus sera were 98.8% (80 of 81) and 100% (10 of 10) at the NIH and STI, respectively; while specificity with Loa loa sera at the NIH was 84.6% (44 of 52). At the STI, the dipstick test also demonstrated 100% specificity when tested with 75 sera from various protozoan and helminthic infections.
Currently, the laboratory diagnosis of toxocariasis, caused by Toxocara canis or T. cati, mainly relies on serological tests. Unfortunately, however, the specificities of most of the commercial tests that are available for the serodiagnosis of this disease are not very high and this may cause problems, especially in tropical countries where co-infections with other helminths are common. In an effort to develop a serological assay with improved specificity for the detection of Toxocara infection, an IgG(4)-ELISA based on a recombinant version (rTES-30USM) of the 30-kDa Toxocara excretory-secretory antigen (TES-30) has recently been developed. To produce the antigen, the TES-30 gene was cloned via assembly PCR, subcloned into a His-tagged prokaryotic expression vector, and purified by affinity chromatography using Ni(2+)-nitrilotriacetic-acid (Ni-NTA) resin. The performance of the ELISA based on the recombinant antigen was then compared with that of commercial kit, based on an IgG-ELISA, for the serodiagnosis of toxocariasis (Toxocara IgG-ELISA; Cypress Diagnostics, Langdorp, Belgium). Both assays were used to test 338 serum samples, including 26 samples from probable cases of toxocariasis. Assuming that all the probable cases were true cases, the assay based on rTES-30USM demonstrated a sensitivity of 92.3% (24/26) and a specificity of 89.6% (103/115) whereas the commercial kit exhibited a sensitivity of 100% (26/26) but a specificity of only 55.7% (64/115). The high sensitivity and specificity exhibited by the new IgG(4)-ELISA should make the assay a good choice for use in tropical countries and any other area where potentially cross-reactive helminthic infections are common.
In Peninsular Malaysia, only Wuchereria bancrofti and Brugia malayi are reported to cause human filariasis. Brugia pahangi infects many of the same animal hosts as the zoonotically transmitted subperiodic B. malayi. There is a well-recognized need for improved diagnostic techniques for lymphatic filariasis. Parasite antigen detection is a promising new approach, and it will probably prove to be more sensitive and specific than clinical, microscopic and antibody-based serological methods. We recently generated monoclonal antibodies (MAb XC3) from in vitro culture products of adult B. pahangi (B.p. IVP). Filarial antigenemia was quantitated in various hosts including the sera from 6 Malaysian Aborigines with acute lymphatic filariasis. In hosts infected with brugian filariasis and dirofilariasis, antigenemia was scored ranging from 90 ng/ml to 960 ng/ml. None of the control animal and human sera had antigenemia above 90 ng/ml. In addition, MAb XC3 and B.p. IVP were applied in several seroepidemiological surveys among household cats in Kuala Selangor in order to correlate information gathered for future studies of possible cases of human infection. Out of the 81 cats surveyed, 10 (12.35%) and 5 (6.17%) were parasitologically positive for B. pahangi and B. malayi, respectively. However, 21 (25.92%) were antigenemia positive when serologically investigated with MAb XC3. Antifilarial antibodies to B.p. IVP by direct ELISA showed very high cross-reactivity with non-filarial gut worm infections. 16 (19.75%) cats had reciprocal titers ranging from 320 to 2,560. Only 1 (1.23%) cat from this group was antigenemic.