Detection and quantification of enteropathogens in stool specimens is useful for diagnosing the cause of diarrhea but is technically challenging. Here we evaluate several important determinants of quantification: specimen collection, nucleic acid extraction, and extraction and amplification efficiency. First, we evaluate the molecular detection and quantification of pathogens in rectal swabs versus stool, using paired flocked rectal swabs and whole stool collected from 129 children hospitalized with diarrhea in Tanzania. Swabs generally yielded a higher quantification cycle (Cq) (average 29.7, standard deviation 3.5 vs. 25.3 ± 2.9 from stool, P<0.001) but were still able to detect 80% of pathogens with a Cq < 30 in stool. Second, a simplified total nucleic acid (TNA) extraction procedure was compared to separate DNA and RNA extractions and showed 92% (318/344) sensitivity and 98% (951/968) specificity, with no difference in Cq value for the positive results (ΔCq(DNA+RNA-TNA) = -0.01 ± 1.17, P = 0.972, N = 318). Third, we devised a quantification scheme that adjusts pathogen quantity to the specimen’s extraction and amplification efficiency, and show that this better estimates the quantity of spiked specimens than the raw target Cq. In sum, these methods for enteropathogen quantification, stool sample collection, and nucleic acid extraction will be useful for laboratories studying enteric disease.

During a scrub typhus outbreak investigation in Thailand, 4 isolates of O. tsutsugamushi were obtained and established in culture. Phylogenetic analysis based on the 56-kDa type-specific antigen gene demonstrated that the isolates fell into 4 genetic clusters, 3 of which had been previously reported and 1 that represents a new genotype.

Scrub typhus, caused by antigenically disparate isolates of Orientia tsutsugamushi, is a widely distributed mite-borne human disease in the Asia Pacific region. Information regarding the heterogeneity of the immunodominant 56-kDa type-specific antigen (TSA) gene is crucial for the design and evaluation of scrub typhusspecific diagnostic assays and vaccines. Using indirect immunofluorescence assays (IFA) and PCR assays, O. tsutsugamushi was detected samples from rodents and patients with fever of unknown origin obtained from six provinces of Thailand during 2004 to 2007. Sequences were determined for a fragment of the 56-kDa TSA gene, and the relationship between these sequences and those previously determined were assessed. The phylogenetic analyses of partial 56-kDa TSA gene sequences demonstrated wide diversity and distribution of O. tsutsugamushi genotypes in Thailand. Furthermore, the genetic diversity grouped the scrub typhus agents into two commonly and five infrequently found genotypes within six provinces of Thailand. The two most commonly found genotypes of O. tsutsugamushi described in this study do not associate with the prototype strains that are widely used for the design and evaluation of diagnostic assays and vaccine candidates. Thus, these new genotypes should be considered for future scrub typhus assay and vaccine development.

Abstract. Orientia tsutsugamushi , an obligate intracellular Gram-negative bacterium, is the causative agent of scrub typhus, a vector-borne disease transmitted by infected chiggers (trombiculid mite larvae). In 2002, an outbreak of scrub typhus occurred among Royal Thai Army troops during the annual field training at a military base in Bothong district, Chonburi province, central Thailand. This report describes the outbreak investigation including its transmission cycle. Results showed that 33.9% of 174 trained troops had scrub typhus-like signs and symptoms and 9.8% of those were positive for O. tsutsugamushi -specific antibodies by indirect fluorescence antibody assay. One hundred thirty-five rodents were captured from this training area, 43% of them had antibodies against O. tsutsugamushi . Six new O. tsutsugamushi isolates were obtained from captured rodent tissues and successfully established in cell culture. Phylogenetic studies showed that these six isolates were either unique or related to a native genotype of previously described isolates from Thailand.

Merozoite surface protein 1 (MSP1) is the major protein on the surface of the plasmodial merozoite, and its carboxy terminus, the 19-kDa fragment (MSP1 19 ), is highly conserved and effective in induction of a protective immune response against malaria parasite infection in mice and monkeys. However, the duration of the immune response has not been elucidated.