1.8.4 Diagnosis of Typhoid fever by antibody-in-lymphocyte-supernatant (ALS) method 1.8.4.

1 Early Use of ALS to Assess Post-vaccination/post infection Immune Response

The ALS method is an immunological assay in which antibodies are secreted by B lymphocytes in the supernatant without in vitro antigen stimulation, and the antibody level in the supernatant is measured by immunoassay. This specific, reliable, and accurate assay was developed for the evaluation of fresh antibody production from circulating mucosal B lymphocytes (Chang and Sack, 2001). It is possible to monitor the magnitude of the mucosal B-cells antibody production strength during the course of immunization through ALS assay. Earlier ALS was used to detect specific antibody response after oral vaccination with a killed cholera vaccine in healthy adults {Chang, 2001 #73}. ALS responses that measure transient presence of mucosal lymphocytes in peripheral circulation after intestinal activation have been reported in vaccinees receiving oral attenuated strains of serovar Typhi (Kirkpatrick et al., 2005; Kirkpatrick et al., 2006; Lundgren et al., 2009). Post-vaccination immunity is generally assayed via the use of antibodies in serum or plasma. But it is difficult to distinguish between recently produced antibodies and preexisting antibodies. The ALS assay is specifically useful for determining recent immune response during vaccine trials in endemic areas where the population already has preexisting serum titers (Chang and Sack, 2001) {Qadri, 2003 #82}. By assaying only antibodies secreted by circulating B lymphocytes, the ALS method controlled the confounding effect of accumulative antibody in the serum samples, which contain both recent and preexisting soluble antibodies. Since the serum portion of blood sample is removed in ALS assay, this assay measures only the actively secreting antibodies. In a study conducted by Chang and Sack, it has been found that antibody titers from pre-vaccination samples were barely detectable in ALS assay, but background titers in serum were found.

Recent antigen exposure of mucosal T and B cells induces proliferation and differentiation of these cells (Hornquist and Lycke, 1995; Vajdy and Lycke, 1993). Activated T and B cells circulate through the thoracic duct into the blood, then return to common mucosal sites, as matured plasma cells (Brenner et al., 1983; Nishibuchi and Seidler, 1983; Rahman et al., 1992; Suthienkul, 1993; Thekdi et al., 1990; Yamamoto et al., 1993). This migration peaks 1 to 2 weeks after intestinal infection. At this time their activity may be measured by using supernatants recovered from ALS assay (Chang and Sack, 2001; Qadri et al., 2003).

1.8.4.2 Application of ALS method as a Diagnostic tool

ALS method can also be used as a diagnostic method for identifying recent infections (Chang and Sack, 2001) . ALS- immunoglobin A (IgA) level has been found to be significantly higher in naturally infected acute cholera patients {Qadri, 2003 #82}. The use of the ALS specimens with the standard ELISA technique holds potential as a future TB-specific diagnostic test (Raqib et al., 2003). With the extensive availability of ELISA technology in developing countries, ALS may be applicable both in developing countries, as well as in industrialized countries. Antibody production of lymphocytes requires multiple signals and optimal cognitive interactions, such as receptor engagement between antigen-presenting cells (APC), T cells, and B cells. The isolated PBMC layer from blood samples contains a mixture of these components. So, antibody production is enhanced by cell concentration and incubation time synergistically. A high concentration of cells in a small space enhances the cognitive distance of cell-to-cell interaction. As the efficiency of cell interaction is increased, antibody secretion is also increased exponentially. The ALS assay quantifies the amount of antibody secreted and the strength of the antibody production for a fixed number of PBMC. Sheikh et al have used ALS specimen derived from PBMC which had natural mucosal priming in the gut to measure antibody response against different typhoid antigens. The highest level of ALS response was seen during the early phase of typhoid, and this decreased at late convalescence showing decrease of circulating antigen-specific B cells. IgA responses to serovar Typhi specific antigens were present in the peripheral circulation of many individuals with

documented or highly suspected serovar Typhi infection at the time of presenting for clinical care settings. 100% blood culture-confirmed typhoid patients showed anti MP-IgA response; 100% individuals highly suspected for typhoid also showed presence of anti MP-IgA response and 70% of Widal positive (1:320 Widal titer) patients also showed response. Large-scale evaluations are needed to determine the true usefulness of ALS assay in the diagnosis of typhoid in countries where typhoid is endemic. ALS specimen can be the basis of improved diagnostic point-of-care-assay for typhoid and warrants further evaluation {Alaullah Sheikh, 2009 #115}.