Assessment and Translation of the Antibody-in-Lymphocyte Supernatant (ALS) Assay to Improve the Diagnosis of Enteric Fever in Two Controlled Human Infection Models and an Endemic Area of Nepal

Abstract

New diagnostic tests for enteric fever are urgently needed to assist with timely antimicrobial treatment of patients and to measure the efficacy of prevention measures such as vaccination. In a novel translational approach, here we use two recently developed controlled human infection models (CHIM) of enteric fever to evaluate an antibody-in-lymphocyte supernatant (ALS) assay, which can detect recent IgA antibody production by circulating B cells in ex vivo mononuclear cell culture. We calculated the discriminative ability of the ALS assay to distinguish diagnosed cases in the two CHIM studies in Oxford, prior to evaluating blood culture-confirmed diagnoses of patients presenting with fever to hospital in an endemic areas of Kathmandu, Nepal. Antibody responses to membrane preparations and lipopolysaccharide provided good sensitivity (>90%) for diagnosing systemic infection after oral challenge with Salmonella Typhi or S. Paratyphi A. Assay specificity was moderate (~60%) due to imperfect sensitivity of blood culture as the reference standard and likely unrecognized subclinical infection. These findings were augmented through the translation of the assay into the endemic setting in Nepal. Anti-MP IgA responses again exhibited good sensitivity (86%) but poor specificity (51%) for detecting blood culture-confirmed enteric fever cases (ROC AUC 0.79, 95%CI 0.70-0.88). Patients with anti-MP IgA ALS titers in the upper quartile exhibited a clinical syndrome synonymous with enteric fever. While better reference standards are need to assess enteric fever diagnostics, routine use of this ALS assay could be used to rule out infection and has the potential to double the laboratory detection rate of enteric fever in this setting over blood culture alone.

Keywords: Salmonella Paratyphi A; Salmonella Typhi; antibody-in-lymphocyte supernatant assay; bacteremia; diagnostic test; enteric fever; febrile illness; resource-limited settings.

Figures

Figure 1

Study sample collection for use in evaluating ALS responses as a diagnostic test for enteric fever. (A) Typhoid challenge study (OVG2009/10). Groups of 20 healthy, screened, and enrolled adult participants were challenged with either 1–5 × 103 CFU or 10–50 × 103 CFU S. Typhi Quailes strain in a dose-escalation study. Participants were challenged by oral ingestion at baseline (D0) and monitored by daily clinical review, routine blood chemistry and hematology and blood culture. Typhoid diagnosis (TD) was made based on a priori definitions of having a temperature ≥38°C for ≥12 h (temperature criterion) and/or having a positive blood culture with S. Typhi (blood culture criterion). Samples for measurement of ALS responses were collected at baseline, TD, TD+48 h, and TD+96 h. Antibiotic treatment was initiated at TD or at D14 in all participants not reaching the typhoid diagnosis endpoint. (B) Paratyphoid challenge study (OVG2013/07). Groups of 20 healthy, screened and enrolled adult participants were challenged with either 1–5 × 103CFU or 0.5–1 × 103CFU S. Paratyphi A strain NVGH308 in a dose-reduction study. Participants were challenged by oral ingestion at baseline (D0) and monitored by daily clinical review, routine blood chemistry, and hematology and blood culture. Paratyphoid diagnosis (PD) was made based on a priori definitions of having a temperature ≥38°C for ≥12 h (temperature criterion) and/or having a positive blood culture with S. Paratyphi (blood culture criterion). Samples for measurement of ALS responses were collected at baseline, D4, D7, D10, D14, and D28. Alternatively, in those reaching PD, samples were collected instead at PD, PD+48 h, and PD+96 h. Antibiotic treatment was initiated at PD or at D14 in all participants not reaching the paratyphoid diagnosis endpoint. (C) Nepal field evaluation. Participants were recruited from individuals presenting with fever and an illness clinically compatible with enteric fever to either Patan Hospital or the Civil Hospital in Patan. Blood for culture and extraction of PBMC/ALS was collected at presentation to hospital (D0), after which antibiotic treatment for enteric fever was given. Further samples were collected to measure ALS responses at D7, month 3 (M3), or month 6 (M6).

Figure 2

ALS IgA diagnostic responses to MP, LPS, and flagellin in participants diagnosed with typhoid after challenge. Low dose, 1–5 × 103CFU; High dose, 10–50 × 103CFU. “White” indicates the titer was below the diagnostic cut-off (log100.95, log101.00, and log101.28EUs for the three antigens tested, respectively). “Gray” indicates sample not available.

Figure 3

ALS IgA responses to MPN or LPS in participants challenged with Salmonella Paratyphi A according to challenge dose level and challenge outcome. High dose, 1–5 × 103 CFU; Low dose, 0.5–1 × 103 CFU. nPD, paratyphoid not diagnosed; PD, paratyphoid diagnosed. “White” indicates the titer was below the diagnostic cut-off (10EU). “Gray” indicates sample not available.

Figure 4

Group ALS responses in each assay condition by time point, according to blood culture result.