Altered Patterns of Compositional and Functional Disruption of the Gut Microbiota in Typhoid Fever and Nontyphoidal Febrile Illness.

BACKGROUND: Experimental murine models and human challenge studies of Salmonella Typhi infection have suggested that the gut microbiome plays an important protective role against the development of typhoid fever. Anaerobic bacterial communities have been hypothesized to mediate colonization resistance against Salmonella species by producing short-chain fatty acids, yet the composition and function of the intestinal microbiota in human patients with typhoid fever remain ill defined. METHODS: We prospectively collected fecal samples from 60 febrile patients admitted to Chittagong Medical College Hospital, Bangladesh, with typhoid fever or nontyphoidal febrile illness and from 36 healthy age-matched controls. The collected fecal samples were subjected to 16s rRNA sequencing followed by targeted metabolomics analysis. RESULTS: Patients with typhoid fever displayed compositional and functional disruption of the gut microbiota compared with patients with nontyphoidal febrile illness and healthy controls. Specifically, typhoid fever patients had lower microbiota richness and alpha diversity and a higher prevalence of potentially pathogenic bacterial taxa. In addition, a lower abundance of short-chain fatty acid-producing taxa was seen in typhoid fever patients. The differences between typhoid fever and nontyphoidal febrile illness could not be explained by a loss of colonization resistance after antibiotic treatment, as antibiotic exposure in both groups was similar. CONCLUSIONS: his first report on the composition and function of the gut microbiota in patients with typhoid fever suggests that the restoration of these intestinal commensal microorganisms could be targeted using adjunctive, preventive, or therapeutic strategies.

Expression of intra- and extracellular granzymes in patients with typhoid fever.

BACKGROUND: Typhoid fever, caused by the intracellular pathogen Salmonella (S.) enterica serovar Typhi, remains a major cause of morbidity and mortality worldwide. Granzymes are serine proteases promoting cytotoxic lymphocytes mediated eradication of intracellular pathogens via the induction of cell death and which can also play a role in inflammation. We aimed to characterize the expression of extracellular and intracellular granzymes in patients with typhoid fever and whether the extracellular levels of granzyme correlated with IFN-γ release. METHODS AND PRINCIPAL FINDINGS: We analyzed soluble protein levels of extracellular granzyme A and B in healthy volunteers and patients with confirmed S. Typhi infection on admission and day of discharge, and investigated whether this correlated with interferon (IFN)-γ release, a cytokine significantly expressed in typhoid fever. The intracellular expression of granzyme A, B and K in subsets of lymphocytic cells was determined using flow cytometry. Patients demonstrated a marked increase of extracellular granzyme A and B in acute phase plasma and a correlation of both granzymes with IFN-γ release. In patients, lower plasma levels of granzyme B, but not granzyme A, were found at day of discharge compared to admission, indicating an association of granzyme B with stage of disease. Peripheral blood mononuclear cells of typhoid fever patients had a higher percentage of lymphocytic cells expressing intracellular granzyme A and granzyme B, but not granzyme K, compared to controls. CONCLUSION: The marked increase observed in extra- and intracellular levels of granzyme expression in patients with typhoid fever, and the correlation with stage of disease, suggests a role for granzymes in the host response to this disease.

The diagnostic accuracy of three rapid diagnostic tests for typhoid fever at Chittagong Medical College Hospital, Chittagong, Bangladesh.

OBJECTIVE: To determine the diagnostic accuracy of three rapid diagnostic tests (RDTs) for typhoid fever in febrile hospitalised patients in Bangladesh. METHODS: Febrile adults and children admitted to Chittagong Medical College Hospital, Bangladesh, were investigated with Bact/Alert(®) blood cultures and real-time PCR to detect Salmonella enterica Typhi and Paratyphi A and assays for Rickettsia, leptospirosis and dengue fever. Acute serum samples were examined with the LifeAssay (LA) Test-it™ Typhoid IgM lateral flow assay detecting IgM antibodies against S. Typhi O antigen, CTKBiotech Onsite Typhoid IgG/IgM Combo Rapid-test cassette lateral flow assay detecting IgG and IgM antibodies against S. Typhi O and H antigens and SD Bioline line assay for IgG and IgM antibodies against S. Typhi proteins. RESULTS: In 300 malaria smear-negative febrile patients [median (IQR) age of 13.5 (5-31) years], 34 (11.3%) had confirmed typhoid fever: 19 positive by blood culture for S. Typhi (three blood PCR positive) and 15 blood culture negative but PCR positive for S. Typhi in blood. The respective sensitivity and specificity of the three RDTs in patients using a composite reference standard of blood culture and/or PCR-confirmed typhoid fever were 59% and 61% for LifeAssay, 59% and 74% for the CTK IgM and/or IgG, and 24% and 96% for the SD Bioline RDT IgM and/or IgG. The LifeAssay RDT had a sensitivity of 63% and a specificity of 91% when modified with a positive cut-off of ≥2+ and analysed using a Bayesian latent class model. CONCLUSIONS: These typhoid RDTs demonstrated moderate diagnostic accuracies, and better tests are needed.

Expression and function of S100A8/A9 (calprotectin) in human typhoid fever and the murine Salmonella model.

BACKGROUND: Typhoid fever, caused by the Gram-negative bacterium Salmonella enterica serovar Typhi, is a major cause of community-acquired bacteremia and death worldwide. S100A8 (MRP8) and S100A9 (MRP14) form bioactive antimicrobial heterodimers (calprotectin) that can activate Toll-like receptor 4, promoting lethal, endotoxin-induced shock and multi-organ failure. We aimed to characterize the expression and function of S100A8/A9 in patients with typhoid fever and in a murine invasive Salmonella model. METHODS AND PRINCIPAL FINDINGS: S100A8/A9 protein levels were determined in acute phase plasma or feces from 28 Bangladeshi patients, and convalescent phase plasma from 60 Indonesian patients with blood culture or PCR-confirmed typhoid fever, and compared to 98 healthy control subjects. To functionally characterize the role of S100A8/A9, we challenged wildtype (WT) and S100A9-/- mice with S. Typhimurium and determined bacterial loads and inflammation 2- and 5- days post infection. We further assessed the antimicrobial function of recombinant S100A8/A9 on S. Typhimurium and S. Typhi replication in vitro. Typhoid fever patients demonstrated a marked increase of S100A8/A9 in acute phase plasma and feces and this increases correlated with duration of fever prior to admission. S100A8/A9 directly inhibited the growth of S. Typhimurium and S. Typhi in vitro in a dose and time dependent fashion. WT mice inoculated with S. Typhimurium showed increased levels of S100A8/A9 in both the liver and the systemic compartment but S100A9-/- mice were indistinguishable from WT mice with respect to bacterial growth, survival, and inflammatory responses, as determined by cytokine release, histopathology and organ injury. CONCLUSION: S100A8/A9 is markedly elevated in human typhoid, correlates with duration of fever prior to admission and directly inhibits the growth of S. Typhimurium and S. Typhi in vitro. Despite elevated levels in the murine invasive Salmonella model, S100A8/A9 does not contribute to an effective host response against S. Typhimurium in mice.