Detection of Antibodies Secreted from CirculatingMycobacterium tuberculosis-Specific Plasma Cells in the Diagnosis of Pediatric Tuberculosis

Abstract

Diagnosis of tuberculosis (TB) in children is difficult because symptoms are often nonspecific or absent in infected children, diagnostic specimens are difficult to obtain from younger children, and >50% have negative TB cultures. Thus, there is an urgent need for improved diagnosis of pediatric TB. This study aimed to evaluate the diagnostic value of a new serological method, the ALS (antibodies in lymphocyte supernatant) assay, for the diagnosis of active TB in children with clinically identified TB. The ALS test is based on the concept that antigen-specific plasma cells are present in the circulation only at times of acute infection and not in latency. A cross-sectional study of pediatric patients (age range, 11 to 167 months) who were clinically identified as TB (n ‫؍‬ 58) or non-TB (n ‫؍‬ 16) patients was conducted, and they were monitored for 6 months. Healthy children (n ‫؍‬ 58) were enrolled as controls. Spontaneous release of TB antigen-specific antibodies by in vitro-cultured, unstimulated peripheral blood mononuclear cells was assessed by an enzyme-linked immunosorbent assay using Mycobacterium bovis bacillus Calmette-Guérin ( According to World Health Organization (WHO) estimates, about 1 million children annually develop tuberculosis (TB) worldwide, accounting for about 11% of all TB cases (25,42,48). Bangladesh ranks 6th among the 22 high-TB-burden countries in the world; about 45% of its population is tuberculin positive at the age of 14 years (22, 48). Childhood TB is often not considered a priority by national TB control programs, because children acquire Mycobacterium tuberculosis infections from adults and do not contribute to disease transmission. However, failure to identify and treat TB in children can lead to death in a majority of Ͻ3-year-old children (14, 23).TB-infected children are often asymptomatic, and bacteriologic confirmation is rare, due to the difficulty of obtaining specimens (8, 31). Most pediatric TB cases are diagnosed using a combination of clinical and epidemiological features, which include characteristic chest radiographs (18), reactive tuberculin skin tests (TST), and a history of contact with active TB cases (31, 34); computed tomography and bronchoscopy are performed in industrialized countries (19). In areas where TB is endemic, most of the population acquires TB infection during childhood, and transmission is not restricted to the household (32,46). This situation limits the diagnostic contribution of documented household exposure and positive TST (10,47). Interpretation of TST reactivity is further complicated by the high prevalences of Mycobacterium bovis BCG vaccination and malnutrition. Radiographic changes in children can be quite variable (18). Serodiagnostic methods are generally low in sensitivity and specificity, especially for children, and seroreactivity data for pediatric TB are also limited (3,4,12,20,26,41). Recent studies evaluating multiple antigens by an enzymelinked immunosorbent assay (ELISA) or a multiantigen print immunoassay for the serodetect...

Characterization of Mucosal Immune Responses to Enterotoxigenic Escherichia coli Vaccine Antigens in a Human Challenge Model: Response Profiles after Primary Infection and Homologous Rechallenge with Strain H10407

“…In recent studies, however, ALS assays have been successfully carried out in the pediatric population in the field using a small volume of blood samples (34,35).…”

A new potential biomarker for childhood tuberculosis

“…The ALS assay was 91% sensitive and 87% specific when compared with the clinical diagnostic algorithm for diagnosing TB disease and performed equally well in TB cases <5 years old compared with those ≥5 years 3. Notably, the BCG-specific IgG titres declined after starting appropriate anti-TB treatment.…”

“…The ALS assay detects antibody secretion from in vivo activated plasma B cells that migrate throughout the peripheral circulation in response to TB antigens that are present during active disease but not latent TB infection 3. The ALS methodology for children includes phlebotomy of 3.5 ml of blood in order to isolate 5 million PBMCs; these cells are incubated in tissue culture plates without stimulation for 48–72 h. The supernatant is collected, placed into BCG-coated microtitre plates and IgG responses to BCG are measured by ELISA 1 3. A positive titre is defined as ≥0.350 optical density units, calculated from data on healthy children 3.…”

“…The ALS methodology for children includes phlebotomy of 3.5 ml of blood in order to isolate 5 million PBMCs; these cells are incubated in tissue culture plates without stimulation for 48–72 h. The supernatant is collected, placed into BCG-coated microtitre plates and IgG responses to BCG are measured by ELISA 1 3. A positive titre is defined as ≥0.350 optical density units, calculated from data on healthy children 3. In its present format, the method requires skilled personnel and equipment for PBMC separation from blood samples, sterile cell cultures and ELISA.…”

Laboratory Diagnosis of Tuberculosis- an Update

“…The test is very helpful in TB in children where sputum collection is difficult and not satisfactory. This is a novel diagnostic method in the diagnosis and subsequent monitoring of treatment effectiveness of paediatric TB 8 . It takes only three days to give the result.…”