GC-MS/MS Profiling of Plant Metabolites.

Gas chromatography coupled to electron ionization (EI) quadrupole mass spectrometry (GC-MS) is currently one of the most developed and robust metabolomics technologies. This approach allows for simultaneous measurements of large number of chemically diverse compounds including organic acids, amino acids, sugars, sugar alcohols, aromatic amines, and fatty acids. Untargeted GC-MS profiling based on full scan data acquisition requires complicated raw data processing and sometime provides ambiguous metabolite identifications. Targeted analysis using GC-MS/MS can provide better specificity, increase sensitivity, and simplify data processing and compound identification but wider application of targeted GC-MS/MS approach in metabolomics is hampered by the lack of extensive databases of MRM transitions for non-derivatized and derivatized endogenous metabolites. The focus of this chapter is the automation of GC-MS/MS method development which makes it feasible to develop quantitative methods for several hundred metabolites and use this strategy for plant metabolomics applications.

Typhoid fever in young children in Bangladesh: clinical findings, antibiotic susceptibility pattern and immune responses.

BACKGROUND: Children bear a large burden of typhoid fever caused by Salmonella enterica serotype Typhi (S. Typhi) in endemic areas. However, immune responses and clinical findings in children are not well defined. Here, we describe clinical and immunological characteristics of young children with S. Typhi bacteremia, and antimicrobial susceptibility patterns of isolated strains. METHODS: As a marker of recent infection, we have previously characterized antibody-in-lymphocyte secretion (TPTest) during acute typhoid fever in adults. We similarly assessed membrane preparation (MP) IgA responses in young children at clinical presentation, and then 7-10 days and 21-28 days later. We also assessed plasma IgA, IgG and IgM responses and T cell proliferation responses to MP at these time points. We compared responses in young children (1-5 years) with those seen in older children (6-17 years), adults (18-59 years), and age-matched healthy controls. PRINCIPAL FINDINGS: We found that, compared to age-matched controls patients in all age cohorts had significantly more MP-IgA responses in lymphocyte secretion at clinical presentation, and the values fell in all groups by late convalescence. Similarly, plasma IgA responses in patients were elevated at presentation compared to controls, with acute and convalescent IgA and IgG responses being highest in adults. T cell proliferative responses increased in all age cohorts by late convalescence. Clinical characteristics were similar in all age cohorts, although younger children were more likely to present with loss of appetite, less likely to complain of headache compared to older cohorts, and adults were more likely to have ingested antibiotics. Multi-drug resistant strains were present in approximately 15% of each age cohort, and 97% strains had resistance to nalidixic acid. CONCLUSIONS: This study demonstrates that S. Typhi bacteremia is associated with comparable clinical courses, immunologic responses in various age cohorts, including in young children, and that TPTest can be used as marker of recent typhoid fever, even in young children.

Evaluation of a typhoid/paratyphoid diagnostic assay (TPTest) detecting anti-Salmonella IgA in secretions of peripheral blood lymphocytes in patients in Dhaka, Bangladesh.

BACKGROUND: Rapid and reliable diagnostic assays for enteric (typhoid and paratyphoid) fever are urgently needed. We report the characterization of novel approach utilizing lymphocyte secretions, for diagnosing patients with enteric fever by the TPTest procedure. METHODOLOGY: TPTest detects Salmonella-specific IgA responses in lymphocyte culture supernatant. We utilized TPTest in patients with suspected enteric fever, patients with other illnesses, and healthy controls. We also evaluated simplified modifications of TPTest for adaptation in laboratories with limited facilities and equipment. PRINCIPAL FINDINGS: TPTest was positive in 39 (27 typhoid and 12 paratyphoid A) patients confirmed by blood culture and was negative in 74 healthy individuals. Among 32 individuals with other illnesses, 29 were negative by TPTest. Of 204 individuals with suspected enteric fever who were negative by blood culture, 44 were positive by TPTest and the patients were clinically indistinguishable from patients with confirmed bacteremia, except they were more likely to be under 5 years of age. We evaluated simplifications in TPTest, including showing that lymphocytes could be recovered using lysis buffer or buffy coat method as opposed to centrifugation, that incubation of cells at 37°C did not require supplemental CO2, and that results were available for majority of samples within 24 hours. Positive results by TPTest are transient and revert to negative during convalescence, supporting use of the test in endemic areas. The results can also be read using immunodot blot approach as opposed to ELISA. Since no true gold standard currently exists, we used a number of definitions of true positives and negatives. TPTest had sensitivity of 100% compared to blood culture, and specificity that ranged from 78-97% (73-100, 95% CI), depending on definition of true negative. CONCLUSION: The TPTest is useful for identification of patients with enteric fever in an endemic area, and additional development of simplified TPTest is warranted.