Immunological Diagnosis

Intradermal tests

Intradermal tests performed by several investigators have produced equivocal results. Early studies with purified antigenic extracts of adult Dirofilaria immitis (Sawada antigen) showed high positive rates in W. bancrofti microfilaremic and clinically positive patients.[5] However, it was found that this antigen could not discriminate between patients with and without clinical manifestations. In addition, the Sawada antigen was reported to have cross-reactivity with other filarial infection.[6] Subsequently, other investigators compared the Sawada antigen with W. bancrofti microfilarial and larval extracts and B. malayi infective larval antigen.[7] From these studies, it appears that antigen prepared from human parasites has greater diagnostic utility in intradermal tests.

Antibody detection

A number of serological assays have been developed for the immunodiagnosis of lymphatic filariasis. These include the indirect fluorescent antibody test (IFAT), indirect hemagglutination test (IHA), precipitin techniques, and the enzyme-linked immunosorbent assay (ELISA). Various stages of antigens have been employed in the IFAT. These include frozen sections of adult worm and larvae,[8] papainized microfilaria,1-9-1 and sonicated fragment microfilaria.1-10-1 From these studies, homologous antigens were found to be better than heterologous antigens for antibody detection. The IHA test and precipitin techniques have not been widely used for the diagnosis of filariasis in recent years as previous studies have shown their lack of sensitivity.1-11-1 However, the ELISA is very popular in the diagnostic laboratory because its sensitivity is reported to be equal to that of radioimmunoassay (RIA).

Nevertheless, the methods that have focused on the detection of filarial antibodies in the host generally lacked sufficient sensitivity and specificity to discriminate effectively between past and present filarial infections in humans. The detection of circulating filarial antigens in the peripheral blood would thus provide a more accurate indication of active filarial infection in man than the detection of antibody.

Antigen detection

Antigen detection assays would be especially useful for diagnosing a microfilaremic prepatent and occult infection where classical parasitological tests are not useful. W. bancrofti represents a success to the detection of circulating antigen, in that an ELISA is commercially available for detection of this parasite (Trop-Ag

W. bancrofti ELISA kit, JCU Tropical Biotechnology Pty Ltd, Queensland, Australia). The assay is dependent on a monoclonal antibody, Og4C3, which, curiously, despite being raised against antigens of the bovine parasite Onchocerca gibsoni, detects circulating antigen in serum samples from individuals infected with W. bancrofti, but not Onchocerca volvulus. Negative results were obtained with sera from individuals harboring B. malayi. A second specific assay, which is currently undergoing commercial development, is based on the mAb AD12.1 which recognizes a 200-kDa antigen in the circulation of individuals infected with W. bancrofti which again is of adult worm origin. Both assays have been evaluated as diagnostic tools in a number of studies and can detect circulating antigen in virtually all (94-100%) microfilaria carriers, as antigen levels remain constant throughout the day.

However, antigen detection methods are now recognized to have advantages over other diagnostic methods for W. bancrofti infection. The ELISA format of assays such as the Og4C3-dependent test is not ideal for use in the field. A new rapid assay has therefore been developed (the filariasis card test produced by ICT Diagnostics using mAb AD12.1). This assay takes only 5-15 min to complete, requires no specialized equipment, and gives comparable results to ELISA. Such assays are likely to prove valuable in the global effort to eradicate lymphatic filariasis as a public health issue. The immunochromatographic test (ICT) kit has been validated and used worldwide as an effective tool in detecting infections, especially in low-level microfilariae carriers, which are not usually detectable by conventional night blood examination. The ICT filariasis card test was recently recommended as a rapid screening tool to define the prevalence and distribution of filariasis as part of the global program to eliminate lymphatic fila-riasis. It was found that adult filarial antigen levels persist in microfilariae-negative persons for up to 3 years after treatment.[12]

Monoclonal antibody-based enzyme immunoassays for detecting parasite antigens would therefore be more suitable for field application. Several groups of researchers have successfully used such assay in the diagnosis of lymphatic filariasis. Weil et al.[13] used two monoclonal antibodies to detect D. immitis in a direct sandwich ELISA and to detect W. bancrofti antigens in human sera.

DNA probes

During the 1980s, a substantial effort to isolate and characterize filarial species-specific DNA probes was undertaken. The main objective was the development of a diagnostic system that would allow assessment of the impact of filarial nematode control programs. Later, the adaptation of polymerase chain reaction (PCR) technology and the development of a procedure involving ELISA detection of PCR products permitted the adoption of DNA probe-based methodology from the bench to the field. It is also of interest to note that effective diagnosis, the initial objective of the DNA probe-based research, was not only achieved, but also surpassed in that a deeper knowledge of the epidemiology of filarial disease, as well as filarial phylogenetic relationships, was achieved from this work.[14] PCR development has had a major impact on filarial diagnosis. The species-specific DNA probe sequences referred to above were used to design PCR primers that allowed both specific and sensitive parasite detection. The specificity of amplified DNA was confirmed by hybridization with species-specific oligonucleotides or the full DNA probes. The use of filarial DNA probe-based research has, in addition to benefiting diagnosis, contributed greatly to our knowledge of the epidemiology and pathology of the corresponding human diseases, as well as to genomic structure and evolutionary connections between related species. W. bancrofti PCR detection systems were developed to identify the parasite in mosquitoes.[15]

Polymerase chain reaction assays

Polymerase chain reaction is an in vitro method for amplifying selected nucleic acid (DNA or RNA) sequences by a pair of oligonucleotide primers. The method consists of repetitive cycles of DNA denaturation, primer annealing (hybridized to their complementary sequences), and extension by DNA polymerase. The product of each PCR cycle is complementary to and capable of binding primers, and so the amount of DNA synthesized is doubled in each successive cycle.

The development of PCR-based assays for the diagnosis of many parasitic and nonparasitic infections offers the possibility of improved sensitivity and specificity. These assays were recently developed for diagnosis of infection with the filarial parasite O. volvulus and B. malayi and are more sensitive than parasitologic diagnosis. The recent identification of a W. bancrofti repeated DNA sequence has enabled the development of a PCR-based assay capable of detecting W. bancrofti genomic DNA in human blood, urine, hydrocele fluid, and mosquito vectors such as Aedes polynesiensis and Culex quinquifasciatus.[16]

Recently, a nested PCR is introduced for improvement of specificity and sensitivity of detection, involving two steps of amplification. In the first step, initial pair of primers is used to generate a long sequence that contains the target DNA sequence. Subsequently, a small amount of this product is used in a second step of amplification, which employs primers to the final target DNA. The efficiency of the second round of amplification is enhanced because of the more rapid and more complete denaturation of the first reaction product as compared with total genome. The application of nested PCR has been used for many infectious diseases including parasite, bacteria, and virus. The seminested PCR is the use of two rounds of PCR and by the replacement of one of the two primers from the first round of PCR with a different primer in the second round. This technique was applied for various kinds of detection such as monoclonal B-cell population in plastic-embedded bone marrow biopsies,[17] bacterial diseases,[18] and viral diseases.[19]

The polymerase chain reaction assay was developed for species-specific detection of filarial parasites in the blood samples from infected individuals (B. malayi, W. bancrofti, and Loa loa) and even in the mosquito vectors.[20,21] The 969 nucleotides of the moderately repetitive W. bancrofti sequence have been described as being species-specific. This sequence has a moderate copy number (450-700) and appears to be interspersed within the parasite genome. The use of this region in PCR to detect W. bancrofti has been documented.1-3-1

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook


Post a comment