Development and Validation of an Arthropod Maceration Protocol for Zoonotic Pathogen Detection in Mosquitoes and Fleas Author(s): Genelle F. Harrison, Jessica L. Scheirer and Vanessa R. Melanson Source: Journal of Vector Ecology, 40(1):83-89. Published By: Society for Vector Ecology URL: http://www.bioone.org/doi/full/10.1111/jvec.12136

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Development and validation of an arthropod maceration protocol for zoonotic pathogen detection in mosquitoes and fleas Genelle F. Harrison1, Jessica L. Scheirer2, and Vanessa R. Melanson2 McGill University, 1205 Avenue Docteur Penfield, Montreal, Quebec, H3A 1B1, Canada, Diagnostics and Laboratory Services Department, Entomology Branch, Walter Reed Army Institute of Research, 503 Robert Grant Ave, Silver Spring, MD 20910, U.S.A., [email protected] 1

2

Received 10 June 2014; Accepted 15 September 2014 ABSTRACT: Arthropod-borne diseases remain a pressing international public health concern. While progress has been made in the rapid detection of arthropod-borne pathogens via quantitative real-time (qPCR), or even hand-held detection devices, a simple and robust maceration and nucleic acid extraction method is necessary to implement biosurveillance capabilities. In this study, a comparison of maceration techniques using five types of beads followed by nucleic acid extraction and detection were tested using two morphologically disparate arthropods, the Aedes aegypti mosquito and Xenopsylla spp. flea, to detect the zoonotic diseases dengue virus serotype-1 and Yersinia pestis. Post-maceration nucleic acid extraction was carried out using the 1-2-3 Platinum-Path-Sample-Purification (PPSP) kit followed by qPCR detection using the Joint Biological Agent Identification and Diagnostic System (JBAIDS). We found that the 5mm stainless steel beads added to the beads provided in the PPSP kit were successful in macerating the exoskeleton for both Ae. aegypti and Xenopsylla spp. Replicates in the maceration/extraction/detection protocol were increased in a stepwise fashion until a final 128 replicates were obtained. For dengue virus detection there was a 99% positivity rate and for Y. pestis detection there was a 95% positive detection rate. In the examination of both pathogens, there were no significant differences between qPCR instruments, days ran, time of day ran, or operators. Journal of Vector Ecology 40 (1): 83-89. 2015. Keyword Index: Vector pathogen detection, nucleic acid extraction, biosurveillance.

INTRODUCTION Arthropod-borne diseases have played a significant role in public health since their implication as disease vectors in the 1870s. Dengue, plague, yellow fever, malaria, louse-borne typhus, leishmaniasis, and filariasis have resulted in more human disease and mortality in the 17th through the 20th centuries than all other causes of disease combined (Gubler 1991). Though control efforts targeting the destruction of larval habitats and the use of insecticides have resulted in an initial diminishing transmission of arthropod-borne diseases, a reemergence occurred in the 1970s. The reasons for this reemergence are complex and poorly understood (Reeves 1972) with the diversion of governmental financial support and subsequent loss of a public health infrastructure being implicated as a cause, along with an increase in pathogen and vector resistance to drugs and insecticides (Gubler 1996). Arthtropd-borne diseases are still a serious public health problem and several are also listed among bioterrorism threats, including Yersinia pestis (the causative agent of plague) transmitted by fleas, Francisella tualarensis (the causative agent of tularemia) transmitted by ticks, and Rickettsia prowazekii (the causative agent of epidemic typhus) transmitted by lice (Meselson et al. 2002). The preferred methods for protection for those residing in or traveling to vector and pathogen endemic regions include vaccinations, prophylactic drugs, and the use of repellents. However, fifteen of the top twenty-four pathogens that affect U.S. troops, a population that is frequently in field conditions, have neither a prophylactic drug or vaccine developed, which leaves the use of repellents and pest management as the only available

options for reducing transmission (Coleman et al. 2009). These protective measures can be made more efficient and less resource intensive by targeting high-risk temporal and geographic regions to implement bio-surveillance for agents of disease. With the contemporary advances in biotechnology, rapid, high-throughput biosurveillance is now possible even in field conditions. Quantitative real-time PCR (qPCR) assays are available for the detection of many arthropod-borne pathogens to include Bunyaviridae, Nairovirus, the causative agent of Crimean Congo Hemorrhagic Fever (Garrison et al. 2007), Alphavirus, the causative agent of Chikungunya fever (Pastorino et al. 2005), Rickettsia typhi and Rickettsia felis, the causative agents of murine typhus and flea-borne spotted fever (Henry et al. 2006), Leishmania spp., the causative agent of leishmaniasis (Wortman et al. 2005), and Plasmodium falciparum and P. vivax, the causative agents of malaria (Monbrison et al. 2003). Hand-held detection assays such as Arthropod Vector Rapid Detection Devices (AV-RDDs) have been or are currently being developed for the detection of dengue virus, Alphavirus, Leishmania spp., P. falciparum and P. vivax, and Flavivirus, the causative agent of Japanese encephalitis (Higgins et al. 2003). Given the capabilities of detection assays, one of the major hurdles for effective biosurveillance hinges on a successful nucleic acid extraction. Obtaining pure, high quality nucleic acid is often confounded in arthropods by the exoskeleton for two reasons. First, the exoskeleton can form a barrier between the tissues and organs in which the pathogen resides unless it is thoroughly macerated. Second, the calcium in the exoskeleton can inhibit qPCR amplification if the nucleic acid extraction method is inadequate (Lardeux et al. 2008). For these reasons, the Diagnostics and Laboratory Services

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Department, part of the Entomology Branch, at the Walter Reed Army Institute of Research (WRAIR), Silver Spring, MD, has developed and validated an arthropod maceration/nucleic acid extraction/pathogen detection protocol for two morphologically different arthropods vectors of two zoonotically disparate pathogens. These pathogens include dengue virus propagated in Aedes aegypti and the Y. pestis bacteria propagated in Xenopsylla spp. fleas, which is ranked among the top bio threat agents pertinent to public health preparedness (Meselson et al. 2002). Pathogen detection was carried out on the Joint Biological Agent Identification and Diagnostic System (JBAIDS), a rugged and field-tested qPCR platform. Herein we describe the development and validation of the maceration and nucleic acid extraction method for mosquitoes and fleas with vector pathogen detection for dengue viral RNA and Y. pestis DNA on the JBAIDS platform. MATERIALS AND METHODS The development of a maceration/extraction/detection protocol involved the following steps for both mosquitoes and fleas. First, five different bead types were compared to identify the material of bead that would most successfully macerate the exoskeleton. Three sample replicates were used per bead type. To complete this, samples were processed in pools containing ten arthropods. Second, nucleic acid was extracted using a 1-2-3 Platinum Path Sample Purification (PPSP) kit (BioFire Diagnostics, Inc., Salt Lake City, UT) and its quality was measured. Third, the developed maceration and extraction method was tested for pathogen detection ability. This was accomplished by first testing one infected arthropod in a proof of concept study (ten replicates), then creating pools of ten arthropods, with one being infected and nine being uninfected. The number of replicates of these pools started at 15 and was increased to 32 replicates to ensure the positive pathogen detection rate remained constant with upscaling. Finally, the developed maceration/extraction method was tested using an intermediate precision study of 128 replicates divided evenly between two operators processing samples on different days, at different times of day (morning vs afternoon), and on different JBAIDS instruments to ensure a robustness in the results given environmental factors. Obtaining infected arthropods Uninfected and infected Ae. aegypti were obtained from the insectary at the WRAIR. Mosquitoes were infected with dengue virus (serotype 1, strain FST 2407, isolated from Iquitos, Peru) via intrathoracic inoculation and were allowed to incubate for ten days at 28° C with a 12:12 L:D cycle. Mosquitoes that did not survive the incubation period were removed to avoid confounding results. The remaining mosquitoes were euthanized by placing the containers at 20° C for 30 min. Mosquitoes were then removed from the transfer containers, distributed to 50 ml Eppendorf tubes, and stored at -80° C until required for the study. Y. pestis (attenuated strain KIM6+ lacking the pCD1 virulence plasmid) infected and uninfected Xenopsylla spp. (fleas) were kindly provided for this study by the Centers for Disease Prevention and Control (CDC) Division of Vector-borne Diseases (Fort Collins, CO). Fleas were received post-euthanasia and kept at -80° C until their use in the study.

June 2015

Arthropod maceration protocol During the protocol development and optimization, a total of five bead types were tested and compared with three goals. The first goal was to examine each bead type’s ability to macerate the arthropod exoskeleton exposing target organs or tissues that typically harbor pathogens. For example, when Xenopsylla spp. ingest a blood meal, the bacterium, Y. pestis, enters the gut, colonizes, blocks the mid-gut, and is then regurgitated when the flea takes a subsequent blood meal. Therefore, to detect if the bacterium was present in the fleas, it was of great importance that the maceration process was able to break apart the abdomen. The second goal was to obtain an extraction of nucleic acid that was both pure (free of proteins and enzymes) and of an adequate concentration. The third goal was to create as much uniformity between protocols as possible when testing for different types of arthropod-borne pathogens in order to make training, preparation, and implementation of techniques in the field as simple as possible. The five bead types selected for analysis included the following: the beads provided in the 1-2-3 Platinum Path Sample Purification (PPSP) kit without the addition of other bead types; the addition of two 5 mm stainless steel beads (QIAGEN, Germantown, MD) to the beads provided in the PPSP kit; the addition of two 3 mm tungsten carbide beads (QIAGEN) to the beads provided in the PPSP kit; the addition of one 5mm glass bead (Sigma-Aldrich, St. Louis, MO) to the beads provided in the PPSP kit; and the addition of two 2 mm zirconium beads (Glen Mills, Inc., Clifton, NJ) to the beads provided in the PPSP kit. Each bead type was tested in triplicate with ten uninfected arthropods per replicate. Total maceration was examined and photographed using a dissecting microscope. Nucleic extraction protocol The protocols for nucleic acid extraction differed for RNA and DNA. For RNA pathogen detection, a powdered antifoam (included in the PPSP kit), beads, 800 µl 1x phosphate-buffered saline (PBS) (Mediatech, Inc., Manassas, VA), 200 µl FLOW protease (included in the PPSP kit), and arthropods were combined in each tube. For DNA extraction, powdered antifoam, 400 µl 1x PBS, 800 µl MB binding buffer (included in the PPSP kit), 20 µl VIBE protease (included in the PPSP kit), and arthropods were combined in each tube. Tubes were capped, inverted three times, and incubated at room temperature for 5 min. After the incubation period, the tubes were transferred to a Disrupter Genie vortex (Vortex-Genie® 2, Scientific Industries, Inc., Bohemia, NY) and macerated for 5 min. Once samples were digested and macerated, nucleic acid extraction was performed using the PPSP kit according to the manufacture’s protocol starting at the “Bind Nucleic Acids to Magnetic Beads” section in the Main Protocol found in the instruction booklet. An 1-M magnetic wand (BioControl PickPen®, Sunrise Scientific, San Diego, CA) was used to transfer samples between the wells. Nucleic acid concentrations and the 260/280 ratio was obtained for the protocol development and optimization portion of this study via a NanoDrop® ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA).

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Detection assays JBAIDS lyophilized PCR reagents were used for both targets (dengue viral RNA and Y. pestis) and were prepared according to the manufacturer’s instructions. The use of lyophilized reagents negates the need for refrigeration, which can be difficult to come by in the field. In brief, the lyophilized reagents were reconstituted using 20 µl of purified nucleic acid and 20 µl reconstitution buffer. Negative controls were reconstituted using 20 µl of molecular biology grade water and 20 µl of reconstitution buffer. Following reagent reconstitution, the PCR reaction mix was evenly divided (~19 µl in each) between duplicate capillary reaction tubes (Roche, Indianapolis, IN). The qPCR amplification was performed on the JBAIDS instrument (software version 3.5.0.72) using the standard JBAIDS RNA protocol: 40° C for 30 min, 94° C for two min, followed by 45 cycles of 94° C for 1 s , and 60° C for 20 s. This standard RNA protocol was employed for both dengue viral RNA and Y. pestis detection reactions. Although, the nucleic acid isolated from Y. pestis is DNA, a standard RNA amplification was performed since field-testing often requires DNA and RNA amplification to be performed simultaneously. RESULTS Bead-comparison maceration results For Xenopsylla spp., the analysis of the purified nucleic acid (total RNA and DNA) revealed that none of the bead types were more successful than the other at producing pure DNA at an adequate concentration (Table 1). However, microscopic inspection revealed that the beads included in the PPSP kit with the addition of two 5 mm stainless steel beads were the only combination able to fully macerate the exoskeleton. The four alternative bead types only managed to remove the appendages (Figure 1). The A260/A280 ratio results suggested that some residual protein was carried over from the nucleic acid extraction process; however, this residual protein did not inhibit the qPCR reaction performed in the proof of concept study (Table 2). Hence, the PPSP kit beads with the additional two 5 mm stainless steel beads were selected since this bead combination was capable of fully macerating the arthropods and producing an adequately pure and concentrated nucleic acid sample. For the Ae. aegypti experiment, the stainless steel, tungsten carbide, and glass beads, when added to the beads provided in the PPSP kit successfully macerated the mosquitoes as seen in the microscopic examination (Figure 2). Each bead had a 260/280 ratio between 1.8 and 2.0 (Table 3) and an acceptable concentration of nucleic acid. To obtain the goal of uniformity between arthropodborne pathogen detection protocols, the stainless steel beads were selected, given that there were no explicit benefits to one bead type over another in mosquitoes. Proof of concept and replicate scale-up When testing individual fleas in the proof of concept study, Y. pestis was detected in all ten of the replicates for 100% effectiveness. The average CP value was 26.3 (SD 4.01), and control qPCR reactions were valid with positive and negative controls giving their expected results (Table 2). This demonstrates that the qPCR reactions were not inhibited by the trace amounts of protein left in the samples post-extraction or the potential residual

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stainless steel from the beads. For Ae. aegypti, dengue viral RNA was also detected in all ten replicates when a single mosquito was tested during the proof of concept study, demonstrating 100% effectiveness. The average CP value was 27.3 (SD 1.10) and assay controls produced the expected results (Table 4). During two scale-up studies, first Y. pestis was detected in 15 out of 15 replicates containing one infected flea to nine uninfected fleas with an average CP of 26.5 (SD 4.80). Then the replicates were increased to 32, and Y. pestis was detected in all 32 replicates with an average CP of 26.7 (SD 2.73). All control samples produced the expected results and were valid (Table 2). When dengue viral RNA was targeted in Ae. aegypti, the first 15 replicates containing one infected to nine uninfected mosquitoes tested positive with an average CP of 29.7 (SD 1.39). Then, when 32 replicates were tested, dengue viral RNA was detected in all replicates with an average CP of 30.3 (SD 2.17). All positive and negative controls produced the expected results (Table 4). Intermediate precision Tables 5 and 6 summarize the data obtained from the Y. pestis detection in Xenospylla spp. and dengue virus detection in Ae. aegypti, respectively. For Y. pestis detection, there was no significant difference between the two JBAIDS instruments (P>0.984), operators (P>0.194), days 1-4 (P>0.423), or morning and afternoon runs (P>0.074) (Table 5). For dengue viral RNA detection in Ae. aegypti, there was no significant difference between the two JBAIDS instruments (P>0.412), operators (P>0.977), days 1-4 (P>0.083), or morning and afternoon runs (P>0.062) (Table 6). Assay controls were valid for all runs performed during this portion of the study. For the detection of Y. pestis DNA, 5.4% of replicates tested negative with three replicates giving split results. For the detection of dengue viral RNA, 2.7% of the replicates tested negative, and one replicate produced a split read. Split results were considered to be positive as long as the negative control was negative because it indicates a low level presence of a pathogen. Though there were negatives in both Y. pestis and dengue virus detection, they are most likely due to the random selection of uninfected arthropods rather than a procedural failure. The Y. pestis infection rate in Xenospylla spp. was assumed to be 95% given that all were known to have consumed a blood meal, but the actual infection rate of these fleas is unknown. The dengue viral infection rate in Ae. aegypti was determined to be approximately 95%, which was confirmed with conventional PCR analysis. DISCUSSION The main goal of this study was to develop and validate an arthropod maceration, extraction, and pathogen detection protocol that could be used for biosurveillance in regions with endemic arthropod vectors. This method should be simple, field ready, and have robust capabilities (Coleman et al. 2009). Overall, the results from this study demonstrate that the maceration protocol is effective in the maceration of two morphologically distinct arthropod vectors, which could potentially be extended to other vectors and pathogens. The PPSP kit coupled with the JBAIDS platform and lyophilized reagents provides the technology that can be used in field conditions with limited need for electricity and no need for the refrigeration of reagents. For this reason, these

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Table 1. Nucleic extraction concentration and purity measurements for Xenopsylla spp. Nucleic Acid Conc. (ng/µl)

Nucleic Acid Conc. SD*

A260/A280 Ratio

A260/A280 Ratio SD*

17.4

0.48

1.13

0.08

Tungsten Carbide (Two 3mm)

26.9

8.01

1.20

0.07

Glass (One 5mm)

25.1

1.72

1.22

0.03

**Stainless Steel (Two 5mm)

26.1

2.36

1.22

0.03

Zirconium (Two 2mm)

19.0

1.94

1.15

0.09

Bead Type PPSP Kit Beads Only PPSP Kit Beads Plus:

*SD – standard deviation. **Bead type selected since it was the only one to completely macerate the Xenopsylla spp. exoskeleton for pathogen detection.

Table 2. Xenopsylla spp. and Y. pestis detection results for proof of concept and scale-up studies. Infected : Uninfected Arthropods

# of Sample Pools Tested

Positive Results

Negative Results

Split Results

Proof of Concept

1:0

10

10

0

Scale-Up 1

1:9

15

15

Scale-Up 2

1:9

32

32

Study

CP** Average

*SD

0

26.3

4.01

0

0

25.0

3.05

0

0

26.7

2.73

*SD – standard deviation. **Cp – same as Ct value, just a different notation due to company instrument variation.

Table 3. Nucleic extraction concentration and purity measurements for Aedes aegypti. Nucleic Acid Conc. (ng/µl)

Nucleic Acid Conc. SD*

A260/A280 Ratio

A260/A280 Ratio SD*

24.6

4.36

1.65

0.09

Tungsten Carbide (Two 3mm)

70.8

25.2

1.87

0.03

Glass (One 5mm)

49.3

2.20

1.85

0.03

**Stainless Steel (Two 5mm)

96.2

31.7

1.94

0.03

Zirconium (Two 2mm) 76.1 *SD – standard deviation. **Bead type chosen for remaining studies.

17.3

1.92

0.07

Bead Type PPSP Kit Beads Only PPSP Kit Beads Plus:

Table 4. Dengue virus detection results for proof of concept and scale-up studies for Aedes aegypti. Infected : Uninfected Arthropods

# of Sample Pools Tested

Positive Results

Negative Results

Split Results

Proof of Concept

1:0

10

10

0

Scale-Up 1

1:9

15

15

Scale-Up 2

1:9

32

32

Study

CP** Average

*SD

0

27.3

1.10

0

0

29.7

1.39

0

0

30.3

2.17

*SD – standard deviation. **Cp – same as Ct value, just a different notation due to company instrument variation.

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Figure 1. Microscopic results for Xenopsylla spp. A) Unmacerated control; B) PPSP kit medium beads only; C) Zirconium beads with PPSP kit medium beads; D) Tungsten carbide beads with PPSP kit medium beads; E) Glass bead with PPSP kit medium beads; F) Stainless steel beads with PPSP kit medium beads; G) Close-up of PPSP kit medium beads only treated fleas reveals an intact exoskeleton; and H) Close-up of PPSP kit medium beads with the addition of the stainless steel beads reveals a fully macerated exoskeleton. arthropod maceration protocols were developed using items that are included with the JBAIDS instrument, such as the PPSP kits and the JBAIDS qPCR lyophilized reagents. Macerating arthropods in pools increases the number that can be processed at one time (300 arthropods with one positive and one negative control). Given the 260/280 data, the maceration protocol described herein should be capable of being used with any nucleic acid extraction method such as CTAB or Qiagen kits, and subsequent qPCR platforms. Results obtained during this validation show that the maceration protocols for fleas and mosquitoes are effective in a laboratory setting where the infection rate can be controlled. Consequently, field testing would be the next step to determine the effectiveness and field practicality of these maceration protocols. Performing vector-pathogen detection assays in the field is advantageous for real-time vector surveillance as it provides the capability to target management geographically and temporally where infected vectors are present. Since real-time data obtained from vector surveillance performed in the field is more rapidly available than samples that must first be shipped to public health laboratories, this data will help to overcome the two main challenges in global outbreaks: late reporting and response

outbreaks (WHO World Health Report 2007). Quicker results in field vector surveillance are also advantageous in prediction modeling (with the use of such programs as Vector Map http:// www.vectormap.org/) ecological patterns of vector transmission, thereby providing a better understanding of cyclical transmission dynamics. Copyright Statement MAJ Vanessa R. Melanson is a military service member. This work was prepared as part of her official duties. Title 17 U.S.C. § 105 provides that ‘Copyright protection under this title is not available for any work of the United States Government.’ Title 17 U.S.C. § 101 defines a U.S. Government work as a work prepared by military service members or employees of the U.S. Government as part of those persons’ official duties. Acknowledgments We thank Drs. Scott W. Bearden and Kenneth Gage from the CDC National Center for Emerging and Zoonotic Infectious Diseases Division of Vector-Borne Diseases for providing us with

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June 2015

Figure 2. Microscopic results for Aedes aegypti. A) Unmacerated pinned Ae. aegypti; B) PPSP kit medium beads only; C) Zirconium beads with PPSP kit medium beads; D) Stainless steel beads with PPSP kit medium beads; E) Tungsten carbide beads with PPSP kit medium beads; and F) Glass bead with PPSP kit medium beads.

attenuated Y. pestis -infected fleas, Tobin Rowland (WRAIR) for providing us with the dengue-infected and uninfected mosquitoes and for his knowledge and insectary support, Robert Putnack (WRAIR) and his colleagues for proliferation of the dengue viral cultures used in this study, and Judith Stoffer, Walter Reed Biosystems Unit, for capturing the images of the arthropods during the protocol development and optimization portion of this study. This study was funded by the Medical Countermeasures Systems (MCS), formally known as the Chemical Biological Medical Systems (CBMS), SP-1 9R24. Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense. Companies or specific equipment used during this study were selected by the authors and is not an endorsement by the Department of the Army or the Department of Defense. REFERENCES CITED Coleman, R.E., L.P. Hochberg, J.L. Putnam, K.I. Swanson, J.S. Lee, J.C. McAvin, A.S. Chan, M.L. Oguinn, J.R. Ryan, R.A. Wirtz,

J.K. Moulton, K. Dave, and M.K. Faulde. 2009. Use of vector diagnostics during military deployments: recent experience in Iraq and Afghanistan. Mil. Med. 174: 904-920. Garrison, A.R., S. Alakbarova, D.A. Kulesh, D. Shezmukhamedove, S. Khodjaev, T.P. Endy, and J. Paragas. 2007. Development of a TaqMan-minor groove binding protein assay for the detection and quantification of Crimean-Congo hemorrhagic fever virus. Am. J. Trop. Med. Hyg. 77: 514-520. Gubler, D.J. 1991. Insects in disease transmission. In: G.T. Strickland (ed). Hunter Tropical Medicine, 7th edition. pp. 9811000. W.B. Saunders, Philadelphia. Gubler, D.J. 1996. The global resurgance of arbovirus disease. Trans. R. Soc. Trop. Med. Hyg. 90: 449-451. Henry, K.M., J. Jiang, P.J. Rozmajzl, A.F. Azad, K.R. Macaluso, and A.L. Richards. 2006. Development of a quantitative realtime PCR assays to detect Rickettsia typhi and Rickettsia felis, the causitive agents of murine typhus and flea-borne spotted fever. Mol. Cell. Probes 21: 17-23. Higgins, J.A., S. Nasarabadi, J.S. Karns, D.R. Shelton, M. Cooper, A. Gbakima, and R.P. Koopman. 2003. A handheld real time thermal cycler for bacterial pathogen detection. Biosens. Bioelectron. 18: 1115-1123. Lardeux, F., R. Tejerina, C. Aliaga, R. Ursic-Bedoya, C.

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Table 5. Xeonpsylla spp. – Y. pestis results from intermediate precision. Operator / Day

Time of Day

Infected: Uninfected Arthropods

# of Sample Pools Tested

Positive Results

Negative Results

Split Results

Op 1 / D1

Morning

1:9

16

12

2

Op 1 / D2

Afternoon

1:9

16

16

Op 1 / D3

Morning

1:9

16

Op 1 / D4

Afternoon

1:9

Op 2 / D1

Afternoon

Op 2 / D2

CP** Average

*SD

2

26.3

3.73

0

0

27.7

2.66

15

1

0

27.2

1.99

16

15

1

0

28.2

3.16

1:9

16

15

1

0

26.7

1.51

Morning

1:9

16

15

0

1

26.9

1.88

Op 2 / D3

Afternoon

1:9

16

15

1

0

29.5

3.07

Op 2 / D4

Morning

1:9

16

15

1

0

28.4

4.07

*SD – standard deviation. **Cp – same as Ct value, just a different notation due to company instrument variation.

Table 6. Dengue virus intermediate precision results for Aedes aegypti. Operator / Day

Time of Day

Infected: Uninfected Arthropods

# of Sample Pools Tested

Positive Results

Negative Results

Split Results

Op 1 / D1

Morning

1:9

16

16

0

Op 1 / D2

Afternoon

1:9

16

16

Op 1 / D3

Morning

1:9

16

Op 1 / D4

Afternoon

1:9

Op 2 / D1

Afternoon

Op 2 / D2 Op 2 / D3

CP** Average

*SD

0

30.5

2.58

0

0

30.6

2.04

16

0

0

28.4

1.85

16

14

2

0

28.5

2.91

1:9

16

16

0

0

28.3

1.52

Morning

1:9

16

16

0

0

29.1

1.87

Afternoon

1:9

16

16

0

0

28.6

1.13

1

29.6

1.20

Op 2 / D4 Morning 1:9 16 15 0 *SD – standard deviation. **Cp – same as Ct value, just a different notation due to company instrument variation.

Lowenberger, and T. Chavez. 2008. Optimization of a seminested multiplex PCR to identify Plasmodium parasites in wild-caught Anopheles in Bolivia, and its application to field epidemiological studies. R. Soc. Trop. Med. and Hyg. 102: 485-492. Meselson, M.J., J. Guillemin, and M. Hugh-Jones. 2002. Public health assessment of potential biological terrorism agents. Emerg. Infect. Dis. 8: 225-230. Monbrison, F.D., C. Angei, A. Staal, K. Kaiser, and S. Picot. 2003. Simultaneous identification of the four human Plasmodium species and quantification of Plasmodium DNA load in human blood by real-time polymerase chain reaction. Am. J. Trop. Med. Hyg. 97: 387-390.

Pastorino, B., M. Bessaud, M. Grandadam, S. Murri, H.J. Tolou, and C.H. Peyrefitte. 2005. Development of a TaqMan RTPCR assay without RNA extraction step for the detection and quantification of African chikungunya viruses. J. Virol. Meth. 124: 65-71. Reeves, W.C. 1972. Recrudescence of Arthopod-borne Diseases in the Americas. Washington: Pan American Health Organization. Scientific Publication (DC), 238. World Health Report. (2007). Chapter 2: Threats to public health and safety. World Health Organization, 19-20. Wortmann, G., L.P. Hochberg, H.H. Houng, C. Sweeney, M. Zapor, N. Aronson, P. Weina, and C.F. Ockenhouse. 2005. Rapid identification of Leishmania complexes by a real-time PCR assay. Am. J. Trop. Med. Hyg.73: 999-1004.

Development and validation of an arthropod maceration protocol for zoonotic pathogen detection in mosquitoes and fleas.

Arthropod-borne diseases remain a pressing international public health concern. While progress has been made in the rapid detection of arthropod-borne...
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