Novel high-throughput screening method using quantitative PCR to determine the antimicrobial susceptibility of Orientia tsutsugamushi clinical isolates.

Objectives: To develop a method to enable the large-scale antimicrobial susceptibility screening of Orientia tsutsugamushi clinical isolates, using one timepoint and one concentration of antibiotics to considerably speed up the time to result. Methods: Growth, harvesting, multiplicity of infection (moi) and the day to determine the MICs were optimized using five O. tsutsugamushi reference strains [susceptible (Karp, Kato and Gilliam) and putatively resistant (AFC-3 and AFSC-4)], one clinical isolate (UT76) and one rodent isolate (TA763). Subsequently, the MICs of azithromycin, chloramphenicol and doxycycline for these strains and 51 clinical isolates including AFSC-7 were determined. An optimal concentration was calculated using the epidemiological cut-off value. Results: The conditions for O. tsutsugamushi infection, growth and harvesting were determined to be an moi of 100:1 and trypsinization with the peak growth on day 10. The resulting MICs were in line with previously published susceptibility data for all reference strains, except for Karp and AFSC-4, which showed azithromycin MICs of 0.0156 and 0.0313 mg/L, compared with 0.0078 and 0.0156 mg/L, respectively, in previous reports. The MIC of doxycycline for AFC-3 was 0.125 mg/L compared with >4 mg/L in earlier reports. The final single screening concentrations were identified as: azithromycin, 0.125 mg/L; chloramphenicol, 8 mg/L; and doxycycline, 1 mg/L. Conclusions: This simplified procedure facilitates the simultaneous screening of 48 isolates for actively monitoring potential resistance of this important fever pathogen, with an 8-fold throughput improvement over early methods. The data do not support the existence of doxycycline- and chloramphenicol-resistant scrub typhus.

Evaluation of a simple blood culture amplification and antigen detection method for diagnosis of Salmonella enterica serovar typhi bacteremia.

In most areas where typhoid is endemic, laboratory diagnosis is not possible due to the lack of appropriate facilities. We investigated whether the combination of blood culture amplification of Salmonella enterica serovar Typhi with an S. Typhi antigen rapid diagnostic test (RDT) could be an accurate and inexpensive tool for the accelerated diagnosis of patients with acute typhoid in Laos. For a panel of 23 Gram-negative reference pathogens, the Standard Diagnostics (catalog no. 15FK20; Kyonggi-do, South Korea) RDT gave positive results for S. Typhi NCTC 8385, S. Typhi NCTC 786 (Vi negative), Salmonella enterica serovar Enteritidis (ATCC 13076), and Salmonella enterica serovar Ndolo NCTC 8700 (all group D). In a prospective study of 6,456 blood culture bottles from 3,028 patients over 15 months, 392 blood culture bottles (6.1%) from 221 (7.3%) patients had Gram-negative rods (GNRs) seen in the blood culture fluid. The sensitivity, negative predictive value, specificity, and positive predictive value were 96.7%, 99.5%, 97.9%, and 87.9%, respectively, for patients with proven S. Typhi bacteremia and 91.2%, 98.4%, 98.9%, and 93.9% for patients with group D Salmonella. The median (range) number of days between diagnosis by RDT and reference assays was 1 (-1 to +2) day for those with confirmed S. Typhi. The use of antigen-based pathogen detection in blood culture fluid may be a useful, relatively rapid, inexpensive, and accurate technique for the identification of important causes of bacteremia in the tropics.