No abstract

19Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by the obligate 20 intracellular Gram-negative bacterium, Anaplasma phagocytophilum. The disease often presents 21 with nonspecific symptoms with negative serology during the acute phase. Direct pathogen 22 detection is the best approach for early confirmatory diagnosis. Over the years, PCR-based 23 molecular detection methods have been developed, but optimal sensitivity is not achieved by 24 conventional PCR while real-time PCR requires expensive and sophisticated instruments. To 25 improve the sensitivity and also develop an assay that can be used in resource-limited areas, an 26 isothermal DNA amplification assay based on recombinase polymerase amplification (RPA) was 27 developed. To do this, we identified a 171-bp DNA sequence within multiple paralogous copies 28 of msp2 within the genome of A. phagocytophilum. Our novel RPA assay targeting this 29 sequence has an analytical limit of detection of one genome equivalent copy of A. 30 phagocytophilum and can reliably detect 125 bacteria/mL in human blood. A high level of 31 specificity was demonstrated by the absence of nonspecific amplification using genomic DNA 32 from human or DNA from other closely-related pathogenic bacteria, such as Anaplasma platys, 33

Carrion's disease is a human disease caused by an infection with Bartonella bacilliformis. Sand fly is believed to be the transmitting vector. Acute infection without treatment is life-threatening with fatality rates as high as 88%. PCR based diagnostic assays have been developed for detecting B. bacilliformis DNA in clinical samples. Genome sequence analysis of B. bacilliformis had identified a segment of 1,162 bp which is present at three different locations. In this study we have developed a qPCR assay targeting this Multiplecopy DNA Segment (MTSeq) to reach a higher sensitivity. The assay sensitivity was evaluated by three different sets of primers. The best set of primers yielded the detection limit of 3.3 bacteria per reaction. DNA extracted from sand flies fed on blood containing B. bacilliformis was also tested. Flies fed on Day 1 and 3 were determined as positive for B. bacilliformis; the results were consistent with the earlier study targeting pap31 gene. The consistency of the qPCR targeting the MTSeq was evaluated using samples containing 8.3 or 3.3 copies of genomic DNA. We demonstrated that 18 out of 36 reactions (50%) were positive for samples containing 8.3 copies of genome; similarly 12 out of 36 reactions (33%) were positive for samples containing 3.3 copies of genome. At the same time, only 8 (25%) and 2 (6%) reactions out of 36 reactions showed positive using primers targeting pap31, respectively. These results have demonstrated that qPCR targeting MTSeq is more sensitive for detecting B. bacilliformis than previous nucleic acid based method targeting pap31.