Optimization of Quantitative PCR Methods for Enteropathogen Detection.

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.

Scrub typhus outbreak, northern Thailand, 2006-2007.

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.

A single injection of 19 kda carboxy-terminal fragment of Plasmodium yoelii merozoite surface protein 1 (PyMSP1(19)) formulated with Montanide ISA and CpG ODN induces protective immune response in mice.

OBJECTIVE: To investigate the efficacy of a vaccine formulation of the 19 kDa conserved carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (PyMSP1(19)) formulated with CpG ODN 1826 and Montanide ISA51 or ISA720 when used to immunize mice by a single injection. METHODS: Groups of BALB/c mice were immunized parenterally with one, two or four injections with PBS or PyMSP1(19) formulated with CpG ODN in ISA51 or ISA720. Sera were collected weekly and assessed for total IgG and IgG subclass titers. Protection was tested by challenge infection with P. yoelii YM. RESULTS: Interestingly, single injection immunization showed the same kinetics of antibody responses as two- or four-injection immunization. However, the peak antibody response induced by PyMSP1(19) in CpG ODN and ISA51 appeared earlier than that induced by PyMSP1(19) in CpG ODN and ISA720 (28 days vs 41 days). At day 63 after the first injection, the PyMSP1(19)-specific IgG antibody levels by single injection and four-injection immunizations were not different. However, the levels of the IgG2a antibody subclass were significantly lower by single injection immunization with PyMSP1(19) in CpG ODN and ISA720. The antibodies were sustained at high levels for at least 20 weeks. After challenge infection, all mice immunized by a single injection of PyMSP1(19) in CpG ODN and ISA51 survived with low-grade parasitemia, while 50% of mice immunized with PyMSP1(19) in CpG ODN and ISA720 died with high levels of parasitemia. CONCLUSION: These findings suggest that MSP1(19) immunization by a single injection can induce protective immunity, particularly when formulated with an appropriate strong adjuvant.

Genotype diversity and distribution of Orientia tsutsugamushi causing scrub typhus in Thailand.

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 typhus-specific 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.

Isolation and characterization of Orientia tsutsugamushi from rodents captured following a scrub typhus outbreak at a military training base, Bothong district, Chonburi province, central Thailand.

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.

Long-lasting protective immune response to the 19-kilodalton carboxy-terminal fragment of Plasmodium yoelii merozoite surface protein 1 in mice.

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. As such, we immunized BALB/c mice with a standard four-dose injection of recombinant Plasmodium yoelii MSP1(19) formulated with Montanide ISA51 and CpG oligodeoxynucleotide (ODN) and monitored the MSP1(19)-specific antibody levels for up to 12 months. The antibody titers persisted constantly over the period of time without significant waning, in contrast to the antibody levels induced by immunization with Freund's adjuvant, where the antibody levels gradually declined to significantly lower levels 12 months after immunization. Investigation of immunoglobulin G (IgG) subclass longevity revealed that only the IgG1 antibody level (Th2 type-driven response) decreased significantly by 6 months, while the IgG2a antibody level (Th1 type-driven response) did not change over the 12 months after immunization, but the boosting effect was seen in the IgG1 antibody responses but not in the IgG2a antibody responses. After challenge infection, all immunized mice survived with negligibly patent parasitemia. These findings suggest that protective immune responses to MSP1(19) following immunization using oil-based Montanide ISA51 and CpG ODN as an adjuvant are very long-lasting and encourage clinical trials for malaria vaccine development.