July – September 2014
Perspectives 1
MG Steve Jones; COL Mustapha Debboun; Richard Burton
The Growing Challenges of Vector...
July – September 2014 The Army Medical Department Center & School PB 8-14-7/8/9
Online issues of the AMEDD Journal are ava...
July – September 2014 1
Perspectives
Commander’s Introduction
MG Steve Jones
Baron Von Steuben introduced Force Health Pr...
2 http://www.cs.amedd.army.mil/amedd_journal.aspx
Editor’s Perspective
As a new century moves through its second decade, ...
July – September 2014 3
THE ARMY MEDICAL DEPARTMENT JOURNAL
Kyushu to update surveillance data and information
from sites...
4 http://www.cs.amedd.army.mil/amedd_journal.aspx
indications were varied and complicated. She was even-
tually diagnosed...
July – September 2014 5
THE ARMY MEDICAL DEPARTMENT JOURNAL
MAJ Colacicco-Mayhugh and her coauthors have pro-
vided a det...
6 http://www.cs.amedd.army.mil/amedd_journal.aspx
Global factors are interacting together to fundamentally
change the wor...
July – September 2014 7
in military preventive medicine and public health as it
relates to infectious disease surveillanc...
8 http://www.cs.amedd.army.mil/amedd_journal.aspx
vector-borne disease morbidity and mortality in these
areas. These real...
July – September 2014 9
THE ARMY MEDICAL DEPARTMENT JOURNAL
and region-specific information, to include cultural
awarenes...
10 http://www.cs.amedd.army.mil/amedd_journal.aspx
2. Colacicco-Mayhugh MG, Gosine S, Hughes T, Di-
claro J, Larson R, D...
July – September 2014 11
Mosquito-borne disease agents can pose a threat to hu-
mans, particularly to deployed troops, bo...
12 http://www.cs.amedd.army.mil/amedd_journal.aspx
for the Anopheles Hyrcanus Group were unclear or
had conflicting infor...
July – September 2014 13
THE ARMY MEDICAL DEPARTMENT JOURNAL
Collected larvae were placed in plastic Whirl-Pak bags
(118 ...
14 http://www.cs.amedd.army.mil/amedd_journal.aspx
MOSQUITO BIOSURVEILLANCE ON KYUSHU ISLAND, JAPAN, WITH EMPHASIS ON
ANO...
July – September 2014 15
THE ARMY MEDICAL DEPARTMENT JOURNAL
other Hyrcanus Group larvae (still to be identified us-
ing ...
16 http://www.cs.amedd.army.mil/amedd_journal.aspx
first described An. yatsushiroensis, provided elaborate
morphological ...
July – September 2014 17
THE ARMY MEDICAL DEPARTMENT JOURNAL
13. Otsuru M, Ohmori Y. Malaria studies in Japan af-
ter Wo...
18 http://www.cs.amedd.army.mil/amedd_journal.aspx
MOSQUITO BIOSURVEILLANCE ON KYUSHU ISLAND, JAPAN, WITH EMPHASIS ON
ANO...
July – September 2014 19
THE ARMY MEDICAL DEPARTMENT JOURNAL
SummaryofcollectionlocalitiesandlarvalhabitatsforAnopheles(A...
20 http://www.cs.amedd.army.mil/amedd_journal.aspx
MOSQUITO BIOSURVEILLANCE ON KYUSHU ISLAND, JAPAN, WITH EMPHASIS ON
ANO...
July – September 2014 21
Vector-borne diseases remain a significant cause of dis-
ease for US service members throughout ...
22 http://www.cs.amedd.army.mil/amedd_journal.aspx
70% ethanol at -80°C. Samples were transferred to
1.5 mL round-bottom ...
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  • 1. July – September 2014 Perspectives 1 MG Steve Jones; COL Mustapha Debboun; Richard Burton The Growing Challenges of Vector-Borne Diseases to Regionally-Aligned Forces 6 COL Leon L. Robert, Jr; COL Mustapha Debboun Mosquito Biosurveillance on Kyushu Island, Japan, with Emphasis on Anopheles 11 Hyrcanus Group and Related Species Leopoldo M. Rueda, PhD; Benedict Pagac; Masashiro Iwakami; et al High-Throughput Vector-Borne Disease Environmental Surveillance by Polymerase 21 Chain Reaction According to International Accreditation Requirements Marty K. Soehnlen, PhD, MPH; CPT Stephen L. Crimmins; Andrew S. Clugston, MS; et al Evaluation of a Rapid Immunodiagnostic Rabies Field Surveillance Test on Samples 27 Collected from Military Operations in Africa, Europe, and the Middle East Kristen M. Voehl, DVM, MPH, DACVPM; LTC Greg A Saturday Trends in Rates of Chronic Obstructive Conditions Among US Military Personnel, 2001-2013 33 Joseph H. Abraham, ScD; Leslie L. Clark, PhD; Jessica M. Sharkey, MPH; Coleen P. Baird, MD, MPH Department of Defense Participation in the Department of Veterans Affairs Airborne 44 Hazards and Open Burn Pit Registry: Process, Guidance to Providers, and Communication Jessica M. Sharkey, MPH; Deanna K. Harkins, MD, MPH; Timothy L. Shickedanz; Coleen P. Baird, MD, PhD Coinfection of Mycoplasma Pneumonia with Chronic Q Fever in a Nurse Deployed to 51 Operation Iraqi Freedom: A Case Study LTC Paul O. Kwon; Jason R. Pickett, MD Over the Ear Tactical Communication and Protection System Use by a 55 Light Infantry (Airborne) Brigade in Afghanistan MAJ Leanne Cleveland Health Hazard Assessment and the Toxicity Clearance Process 59 Mohamed R Mughal, PhD; John Houpt; Timothy A. Kluchinsky, Jr, DrPH Chemical and Biological Warfare: Teaching the Forbidden at a State University 61 CDR (Ret) David M. Claborn, USN; Keith Payne, PhD An Introduction to Public Health Law for Leaders and Clinicians 68 Joseph Baar Topinka, JD, MHA, MBA, LLM The Public Health Specialist Program at the Medical Education and Training Campus 72 MAJ M. G. Colacicco-Mayhugh; LT Carl Blaesing, USN; LTC Kent Broussard Using the Army Medical Cost Avoidance Model to Prioritize Preventive Medicine Initiatives 76 Cindy Smith; Kelsey McCoskey, MS; MAJ Jay Clasing; Timothy A. Kluchinsky, Jr, DrPH Managing Public Health in the Army Through a Standard Community 82 Health Promotion Council Model Anna F. Courie, RN, MS; Moira Shaw Rivera, PhD; Allison Pompey, DrPH, CPH Performance Excellence: Using Lean Six Sigma Tools to Improve the US Army 91 Behavioral Health Surveillance Process, Boost Team Morale, and Maximize Value to Customers and Stakeholders Eren Youmans Watkins, PhD, MPH; Dave M. Kemeter, MBB; Anita Spiess, MSPH; et al FORCE HEALTH PROTECTION: THE PROVEN FORCE MULTIPLIER
  • 2. July – September 2014 The Army Medical Department Center & School PB 8-14-7/8/9 Online issues of the AMEDD Journal are available at http://www.cs.amedd.army.mil/amedd_journal.aspx A Professional Publication of the AMEDD Community The Army Medical Department Journal [ISSN 1524-0436] is published quarterly for The Surgeon General by the AMEDD Journal Office, USAMEDDC&S, AHS CDD 3599WinfieldScott RD STE B0204,JBSAFortSam Houston,TX78234-4669. Articles published in The Army Medical Department Journal are listed and indexed in MEDLINE, the National Library of Medicine’s premier bibliographic database of life sciences and biomedical information. As such, the Journal’s articles are readily accessible to researchers and scholars throughout the global scientific and academic communities. CORRESPONDENCE: Manuscripts, photographs, official unit requests to receive copies, and unit address changes or deletions should be sent via email to usarmy.jbsa.medcom-ameddcs.list.amedd-journal@mail.mil, or by regular mail to the above address. Telephone: (210) 221-6301, DSN 471-6301 DISCLAIMER: The AMEDD Journal presents clinical and nonclinical professional information to expand knowledge of domestic & international military medical issues and technological advances; promote collaborative partnerships among Services, components, Corps, and specialties; convey clinical and health service support information; and provide a peer-reviewed, high quality, print medium to encourage dialogue concerning healthcare initiatives. Appearance or use of a commercial product name in an article published in the AMEDD Journal does not imply endorsement by the US Government. Views expressed are those of the author(s) and do not necessarily reflect official policies or positions of the Department of the Army, Department of the Navy, Department of the Air Force, Department of Defense, nor any other agency of the US Government. The content does not change or supersede information in other US Army Publications. The AMEDD Journal reserves the right to edit all material submitted for publication (see inside back cover). CONTENT: Content of this publication is not copyright protected. Reprinted material must contain acknowledgement to the original author(s) and the AMEDD Journal. OFFICIAL DISTRIBUTION: This publication is targeted to US Army Medical Department units and organizations, other US military medical organizations, and members of the worldwide professional medical community. LTG Patricia D. Horoho The Surgeon General Commander, US Army Medical Command MG Steve Jones Commanding General US Army Medical Department Center & School Administrative Assistant to the Secretary of the Army GERALD B. O’KEEFE 1411106 By Order of the Secretary of the Army: Official: Raymond T. Odierno General, United States Army Chief of Staff DISTRIBUTION: Special
  • 3. July – September 2014 1 Perspectives Commander’s Introduction MG Steve Jones Baron Von Steuben introduced Force Health Protection to the United States Army at Valley Forge when he in- structed regimental commanders that “the preservation of the Soldier’s health should be their first and greatest care.” Captains, lieutenants, and ensigns were given ad- ditional instructions on their role in supporting this new leadership responsibility. The concept has served the Army well since its introduction, and, with improved techniques, we saw significantly higher casualty sur- vival rates and lower disease and nonbattle injury rates during the conflicts in Iraq and Afghanistan. Joint Publication 4-02 defines Force Health Protection as “Measures to promote, improve, or conserve the be- havioral and physical well-being of Service members to enable a healthy and fit force, prevent injury and ill- ness, and protect the force from health hazards.”1(pGL-7) Among other things, an October 31, 2006 change added a chapter on Force Health Protection1(pIV-1) with subor- dinate core functional capabilities, the first of which is Casualty Prevention, which includes all measures taken by commanders, leaders, individual military personnel, and the healthcare system to promote, improve, or con- serve the mental and physical well-being of military per- sonnel. Additional core functional capabilities specified and detailed in Joint Publication 4-02 are Preventive Medicine, Health Surveillance, Combat and Operational Stress Control, Preventive Dentistry, Vision Readiness, and Laboratory Services. In his 1984 article in Military Medicine,2 COL Ron Bel- lamy stimulated critical thinking about our approach to combat casualty care. He stated: Given optimal circumstances, such as in Vietnam, neither the application of sophisticated technologies designed to improve survival of traumatized patients in surgical in- tensive care wards or operating rooms, nor greater suc- cess in managing the common causes of postoperative death—sepsis and multiple organ failure—will have a significant impact on improving combat casualty care. Rather, he insisted that improvement would come with “a renewed emphasis on field medical care.” That renewed emphasis was realized with implementation of the Com- bat Lifesaver Program, the 68W Combat Medic, and pub- lication of Tactical Combat Casualty Care guidelines. We taught Soldiers to apply tourniquets, open airways, and decompress a tension pneumothorax, and issued them the Improved First Aid Kit. Also, we taught tactical lead- ers their role in casualty management and evacuation. The new approach to field medical care together with advances in Force Health Protection contributed to sig- nificantly increased survival rates after wounding on the battlefield over the past 12 years. Improved individual body armor with an increased area of coverage and great- er ballistic protection decreased the incidence of thoracic and abdominal injuries. The flame-resistant Army Com- bat Shirt, Advanced Combat Helmet, and ballistic eye protection also provided additional personal protection. Vehicular armor evolved throughout the conflict from the improvised armor added to vehicles by units early in the war to up-armored HMMWVs* and MRAPs.† Each of these advances enhanced Soldier survivability. While we should continue to seek technological advanc- es such as improved surveillance for biological weap- ons, the human dimension of Force Health Protection deserves further attention. Just as critical thinking about care on the battlefield led to significant improvements in combat casualty care, critical thinking about the human factors that lead to casualties may significantly reduce their numbers. My analysis of casualties as Command Surgeon, Multinational-Force Iraq from 2005-2006 showed that service members were at greater risk of becoming a casualty during their first 90 days in the- ater. When these findings were forwarded up the chain of command, they prompted the Secretary of Defense to question whether shorter but more frequent tours in combat placed warfighters at an increased risk. The Defense Advanced Research Projects Agency sub- sequently commissioned a study to identify critical fa- tality time periods, training, information, and equipment gaps.3,4 Researchers analyzed data on 1,770 US and 215 UK military fatalities in Iraq and Afghanistan from Jan- uary 1, 2007 to September 1, 2009, and interviewed Sol- diers and subject matter experts. They found that nearly 40% of fatalities occurred in the first 3 months of de- ployment, most often attributed to a lack of experience. Force Health Protection and the Human Dimension *High mobility multipurpose wheeled vehicle †Mine resistant ambush protected vehicle
  • 4. 2 http://www.cs.amedd.army.mil/amedd_journal.aspx Editor’s Perspective As a new century moves through its second decade, the world continues to present increasingly perplexing and dangerous challenges for stability and freedom. Threats continually ebb and flow, appearing, disappearing, re- locating, shifting into different forms, and spreading. Military operations planners try their best to anticipate the nature and locations of these threats, and design strategies and doctrine to best counter them. A common thread across all planning is the certainty that future op- erations will continue to encounter serious threats un- related to an anticipated military opponent, especially in undeveloped, remote areas throughout the world. In their article, COL Leon Robert and COL Mustapha Debboun address the variety and breadth of the threat posed by vector-borne diseases and how Army medical planners must tailor their efforts to support the regional- alignment model of building combat capacity and capa- bility. Although the US military has an extensive, world- wide infrastructure involved in disease surveillance and research, experience has shown that there continue to be gaps in data and information about resources for many locations where military operations may be required. This article discusses how those existing capabilities may take advantage of any locally-based resources, and the requirements for planning to work closely with other US and foreign government organizations, as well as nongovernmental organizations which are providing services in the regions. As with the military threats mentioned above, the vectors that carry dangerous pathogens themselves appear, disappear, change, and spread, an increasingly serious problem in this modern world of ubiquitous modes of transportation. Current, accurate data concerning the presence and identification of such vectors in areas of concern are vital to those planning medical support for military operations. An example of the commitment and effort involved in gathering and maintaining such data is presented by Dr Leopoldo Rueda and his colleagues. Their article describes a biosurveillance project targeting mosquito species on the southern Japanese island of PERSPECTIVES Loss of local intelligence when an old unit leaves and a lack of familiarity with the environment and enemy tactics were also cited as contributing factors. A second spike in fatalities occurred at the 6-month point in the tour which Soldiers attributed to complacency. A minor spike in Soldier fatalities noted at the 10-month mark was attributed to fatigue, complacency, and stale tactics. The US Army Human Dimension Concept5 provides a framework to address the human factors of Force Health Protection. A better understanding of the cognitive, physical, and social components may lead to improve- ments in training and better communication of success- ful tactics, techniques, and procedures. Learning, train- ing, repetition, and practice all affect cognition and the decision-making process. Initiatives to accelerate learn- ing and compress the time it takes to accumulate experi- ential competence may shorten the high risk period early in a tour. Physical factors such as fatigue, sleep depriva- tion, dehydration, hunger, and stress from heat or cold affect decision-making in combat. Psychological factors including complacency, stress, boredom, motivation, and a sense of isolation affect decision-making as well. Recognition of these factors and actions to mitigate their effects may reduce casualty rates. Improved physical fit- ness, nutrition, and psychological fitness can help delay the onset of fatigue. A reduction in the use of energy drinks and long hours playing video games after mis- sions will lead to better sleep, and more rested Soldiers. Improved social fitness with better self-discipline, valued relationships, and good communication with others will produce Soldiers who are more resilient and resistant to stress. Application of the Human Dimension Concept has great potential for not only enhancing performance, but also improving health and fitness, and preventing in- jury and illness—the goal of Force Health Protection. References 1. Joint Publication 4-02: Health Service Support. Washington, DC: Office of the Chairman, Joint Chiefs of Staff; July 26, 2012. Available at: http:// www.dtic.mil/doctrine/new_pubs/jp4_02.pdf. Ac- cessed June 11, 2014. 2. Bellamy RF. The causes of death in conventional land warfare: implications for combat casualty care research. Mil Med. 1984;149(2):55-62. 3. Plank T, Scheff S, Sebok A. First 100 days of de- ployment critical to soldier survivability. Na- tional Defense [serial online]. May 2010. Avail- able at: http://www.nationaldefensemagazine.org/ archive/2010/May/Pages/First100DaysofDeploy mentCriticaltoSoldierSurvivability.aspx. Accessed June 11, 2014. 4. Scheff S, Sebok A. Capturing Insights to Reduce Future Warfighter Fatalities [preliminary report]. Washington, DC: Defense Advanced Research Projects Agency; April 2010. Available at: http:// www.manprint.army.mil/documents/2010/Scott_ Scheff.pdf. Accessed June 11, 2014. 5. TRADOC Pamphlet 525-3-7: The U.S. Army Hu- man Dimension Concept. Fort Eustis, VA: US Army Training and Doctrine Command; May 21, 2014. Available at: http://www.tradoc.army.mil/ tpubs/pams/TP525-3-7.pdf. Accessed June 11, 2014.
  • 5. July – September 2014 3 THE ARMY MEDICAL DEPARTMENT JOURNAL Kyushu to update surveillance data and information from sites identified as having taxonomic and ecological importance. The article provides a detailed look at a biosurveillance project: the precollection research to better target collection areas; the multiyear collection effort itself; and the extensive work required to identify the collected specimens and then analyze the results against historical data. Determination of the presence of pathogens among col- lected specimens of potential vectors is the critical step in the surveillance of an area to plan countermeasures against diseases. Such testing can be time consuming, and sometimes can only be performed at a laboratory distant from the area of concern. Dr Marty Soehnlen, CPT Stephen Crimmins, and their colleagues developed a method of standardized testing of surveillance samples using polymerase chain reaction methods which pro- vides high-throughput and allows analysis of multiple pathogens from the same sample. They developed tests for 9 pathogens under international accreditation stan- dards which can be performed at the US Army Public Health Command Region Europe Laboratory Sciences laboratory, but have universal Department of Defense application. Their detailed report should be of great in- terest to all those charged with providing surveillance of vector-borne diseases throughout not only military re- sources, but all other government disease management agencies as well. Throughout most of recorded history, rabies has been known to mankind as a deadly, infectious disease. It has been a target for treatment and elimination from the dawn of medical science, yet today it is still present on every continent except Antarctica. It is found in many species of mammals, both feral and domesticated, in rural and urban environments. Although infections in humans is relatively rare in the United States, rabies poses a con- stant threat to military personnel involved in all types of deployed settings. As such, prompt and reliable iden- tification of its presence is very important to preventive medicine and healthcare delivery personnel. Dr Kristen Voehl and LTC Greg Saturday have contributed an arti- cle describing a research study in which they evaluated a commercially available test kit which can be used in aus- tere settings to confirm rabies infection in brain tissue. The results of their study, combined with that of their lit- erature review, could result in another important tool for medical support units charged with protecting our troops from diseases and other environmental hazards. Respiratory health has received increasing attention from researchers over the last century or so, as the rela- tionship of damage to airborne hazards in the workplace, smoking, and general air pollution has been proven and publicized. Military personnel can be exposed to a complex array of potential hazards and pollutants, sometimes highly concentrated, but more often present in lower concentrations which become an unnoticed, ever-present part of the environment. The Persian Gulf conflicts since 1991 have bought the concerns about respiratory health of Warfighters to an elevated level. Dr Joseph Abraham and his colleagues conducted an extensive, detailed study of medical treatment records concerning chronic obstructive pulmonary disease and associated conditions for active duty military personnel across the 13-year period of current combat operations in the Middle East and Afghanistan. They were seeking information on rates of the conditions, trends, and char- acteristics of the study population reflecting any trends. Their article is a clear description of the extensive data collected, the detailed data reduction performed, and the careful analysis of that data. The data is presented clearly and logically throughout the article. Some of the results were unexpected, others are not readily explain- able. This study is a significant contribution to the body of research into respiratory health of our military per- sonnel, and should generate further investigations. Indicative of the level of concern regarding the respi- ratory health of military personnel who have served in combat theaters is language contained in the Dignified Burial and Other Veterans Benefits Improvement Act of 2012. That language directs the Department of Veterans Affairs to establish a registry to collect information re- garding all potential exposures experienced by military personnel during deployments that might adversely af- fect their respiratory health. The article provided by Jes- sica Sharkey et al presents an excellent description of the purpose and parameters of the registry which is slated to be active before the end of 2014. Their article details the eligibility criteria and registration process for those wishing to enter the registry. There is also important information for military healthcare providers regarding the military healthcare system’s perspective, responsi- bilities, and obligations to those requesting clinical as- sessments as part of the registration. This article is an excellent “heads up” for participants and caregivers alike concerning the impending availability of an important new aspect of healthcare for those who have served. As has been noted in the pages of the AMEDD Jour- nal time and again, military healthcare providers in deployed environments must always “expect the unex- pected” when faced with confusing, conflicting, unfa- miliar diagnostic indications. LTC Paul Kwon and Dr Jason Pickett relate such a situation in their excellent case study of a patient whose symptoms and diagnostic
  • 6. 4 http://www.cs.amedd.army.mil/amedd_journal.aspx indications were varied and complicated. She was even- tually diagnosed as having Q fever, which was present as a coinfection with mycoplasma pneumoniae, a com- bination rarely addressed in medical literature. Further, she had no history of the typical risk factors associated with Q fever during her deployment. This article is a well-organized, detailed, and complete; a superb exam- ple of medical professionalism at its best. Over the last 6 years, the Army Hearing Program has been addressed in several Journal articles, beginning with its conception, its implementation among garrison units, and how it was adopted in the deployed, combat environment. In this issue, MAJ Leanne Cleveland re- turns with an article that provides, among other things, an update on the level of understanding of the importance of hearing protection at the individual Soldier level, and how many have “tested” different hearing devices in combat, sometimes obtained at their personal expense. As part of the returning Soldier health assessments, she conducted a survey of returning troops to quantify their opinions of effectiveness of protective devices, as well as their preferences. The resulting data may be the first concerning protective devices to be collected from those who very recently experienced actual combat rather than a simulated test environment. The results of the surveys were then compared to the pre- and postdeployment au- diograms to determine any correlation between those reporting use of protective devices, and those who did not. The information in this article should be of value to researchers, designers, and testers of protective hearing devices for the military, which will have broad civilian application as well. The safety of the end user has long been a concern for developers of military equipment and weapons. In 1983, the Army formally established the Health Hazard As- sessment Program, which had been operating under The Surgeon General since 1981, with the responsibility to evaluate and monitor development and procurement of all Army materiel systems, including weapons, equip- ment, clothing, etc. As manufacturing processes and the materials used have become more sophisticated and complex, the potential for toxicity in resulting products to have harmful effects on humans and other living things has become an ever more important factor. This is addressed by the Army’s Toxicity Clearance process which is detailed in the article by Dr Mohamed Mughal and his coauthors. They describe the formal investigative and clearance requirements involved for a manufacturer to obtain approval to use specific chemicals and other materials in products before introduction into the Army supply system. This is an interesting look at another im- portant function performed by AMEDD professionals, largely in the background, that is vital to protecting the health of our Soldiers every single day. History is replete with examples of best-intended, con- sensus actions proving fruitless because someone does not follow the plan. Since 1899, a series of international laws, treaties, pronouncements, and unilateral actions have been intended to eliminate chemical and biological weapons, with the inevitable result that those weapons continue to exist under control of the most dangerous of lawless regimes and tyrants. However, since the notion that the problem was solved was conveyed by the coop- erating governments, interest in those weapons waned in both academic and official circles. As a result, people truly knowledgeable in chemical and biological warfare and associated subjects became more difficult to find. A number of events over the last 30 years has exposed and reestablished the vital need for this area of expertise. In their article, CDR (Ret) David Claborn and Dr Keith Payne describe a fellowship and graduate program of study in Countering Weapons of Mass Destruction now offered by Missouri State University in cooperation with the National Defense University in the Washington, DC area. This is an important and very informative article about a proactive approach to dealing with a real-world threat with potentially disastrous ramifications. Sadly, this threat will likely not be truly eliminated any time soon, if ever. Regular readers of the AMEDD Journal are familiar with MAJ (now retired) Joseph Topinka’s contributions, along with those of his colleagues, of excellent articles focused on various legal topics and considerations spe- cifically related to military medicine. For this issue, he has submitted an article providing an overview of public health law. This article is written as an introduction for military medical personnel and other leaders to convey the breadth and depth of legal considerations that per- tain when working within the public health sector. It is an interesting, easy read, and definitely eye-opening as the reader begins to understand how integral and impor- tant a basic understanding of the law is to successfully functioning in the various areas of public health. Among the many changes resulting from the 2005 Base Realignment and Closure Commission recommenda- tions was the consolidation of all enlisted basic and most specialty medical training into a single Medical Education and Training Campus (METC) at Fort Sam Houston, Texas. The METC became fully operational in September 2011. One of the consolidated specialty areas taught at METC is the Public Health Specialist Program, which graduates Army and Navy Preventive Medicine Specialists and Technicians (respectively). PERSPECTIVES
  • 7. July – September 2014 5 THE ARMY MEDICAL DEPARTMENT JOURNAL MAJ Colacicco-Mayhugh and her coauthors have pro- vided a detailed description of that program, and how it has already been revised to optimize both resources and schedule based on initial experience teaching a multi- service curriculum. Their article provides excellent in- sight into the dedication and high level of expertise and professionalism that is involved in the design, delivery, evaluation, and revision cycle necessary to ensure that students receive only the best training possible. The future health and readiness of many military members may directly depend on the knowledge and skills im- parted to these new Soldiers and Sailors. Throughout history, militaries have always faced the Gordian knot of how to satisfy the ever increasing re- quirements their governments/leaders place on them, while simultaneously receiving ever decreasing resourc- es with which to accomplish those tasks. Today’s envi- ronment is no different. Politics, economic factors, shift- ing priorities, new and resurfacing threats, and many other factors result in unpredictable financial resources which must be divided into many parts. In most cases, the amount of available funds is finite, so competition for a share of those funds can be intense. Usually that com- petition hinges on the value to the organization realized from the committed funding, the classic return on in- vestment (ROI). The government data source for typical personnel costs does not cover all of the categories af- fecting the true amount of medical costs, thus degrading the suitability of the data for use in evaluating preven- tive medicine program initiatives. Cindy Smith and her coauthors have provided a detailed description of a tool developed within the Army Institute of Public Health which draws specific, relevant data into analysis to cal- culate a more accurate and realistic ROI for prevention programs. This excellent article is, in essence, a tutorial for use of the analysis tool, clearly explaining the process in a straightforward manner. This powerful analysis tool should be of great interest and utility to all preventive medicine program developers in Army medicine. In recent years, the news coverage of the relief respons- es to large scale disasters have increasingly focused on the apparent disorganization and inability to coordinate within and among government organizations involved in the efforts. While the news organizations overem- phasize these problems for the ratings benefits of hyp- ing the latest, biggest scandal, the basic difficulties that are highlighted in such reports do in fact reflect the complexity of public health systems of virtually every scale, even the seemingly benign and routine function- ing within a military installation. As Anna Courie and her coauthors clearly describe, the public health sys- tems in both the civilian communities and on military installations share the same characteristic of dependence on various, distinct resources and components which are themselves responsible to disparate organizations and authorities. This fragmented structure wastes resources, causes confusion, is error prone, and is obviously slow and ponderous in functioning and reacting to public health issues. The Army has adopted a structure across its installations called the community health promotion council (CPHC) to manage the Army Public Health Sys- tem. The Army Public Health Command has established a standard model for the CPHC to ensure a coordinated approach to managing public health responsibilities and functions. The standard CPHC has been implemented in 12 of the larger Army installation in the United State, while 14 others have CPHC processes but have not as yet received the additional resources necessary to adopt the standard model. Ms Courie et al investigated the rela- tive effectiveness of the public health systems aboard installations with standard CPHC processes versus those without the features of the standard model. Their well-researched article carefully develops the founda- tion for the CPHC approach, and details the results of their study. This is an interesting, informative look at the complexity and broad range of concerns, both tangible and perceptive, that are involved in managing an effec- tive public health system. Lean Six Sigma (LSS) is a well-known process improve- ment, problem-solving methodology that is used by activities within the US Army, including those within AMEDD. Although best known for its success within business and manufacturing, it has also proven valu- able in use with knowledge-based processes. Dr Eren Youmans Watkins and her colleagues applied LSS to a public health surveillance function within the Army’s Behavioral Social Health Outcomes Program, specifi- cally the report of the suicide behavior surveillance data. Their article clearly details the application of the LSS methodology as a carefully designed, step-by-step pro- cess to define the project, measure the existing process parameters, analyze the data, determine the points of substandard performance and the causes, develop and imcorporate changes to improve the processes, and measure and document the resulting performance. For this project, the total number of labor hours (baseline) required to produce each report was 448. Following the project, the number of hours was 199. Also, it was deter- mined that a quarterly report was not seen to be neces- sary by its users, so the report is now produced annually, resulting in additional savings. This article presents an excellent introduction to LSS for those unfamiliar with it. It demonstrates how even a seemingly straightforward process reveals much room for improvement once the components are identified and analyzed in detail.
  • 8. 6 http://www.cs.amedd.army.mil/amedd_journal.aspx Global factors are interacting together to fundamentally change the worldwide vector-borne disease threat and the corresponding public health landscape. These chal- lenges are predicted to become increasingly problematic in the future. Specific challenges such as climate change, rapid urbanization of megacities, lack of clean drink- ing water, poor sanitation and health, and the growing threat of emerging and reemerging vector-borne dis- eases will certainly present an increasing challenge for military entomologists and public health professionals. In addition, these challenges will almost certainly place increasing demands on regionally-aligned US forces in terms of operational requirements, available manpower, and resource consumption. In 2012, the Chief of Staff of the Army announced that future Army forces must be tailored to meet local re- quirements, must be rapidly deployable and scalable from squad to corps level.1 The changing strategic envi- ronment necessitates organizing the Army for regional conditions in an effort to align forces regionally to spe- cific combatant commanders, such as those in Europe, the Pacific, and in Africa. In addition, regionally-aligned forces will require intimate integration with regional al- lies and multinational forces. This overall effort seeks to increase specialized regional expertise and cultural awareness so US forces will be better prepared to meet present and future regional requirements. Through the regional alignment of forces, we will meet both these imperatives, ensuring that our Army remains globally responsive and regionally engaged. As regional alignment of forces begins, Department of Defense (DoD) planners and military forces worldwide continue to face numerous challenges from vector-borne diseases. Not only must military healthcare systems continually adapt to emerging and re-emerging diseases to protect US forces, but they must operate in develop- ing countries that either cannot or will not address con- tinuing high mortality from infectious diseases.2 These countries have little or no surge capacity (ie, numbers of hospital beds, medical supplies, adequate distribu- tion systems, and measures for managing public panic) in case of an influenza pandemic, bioterrorist attack, or even a regional outbreak of a vector-borne disease. Gov- ernments will increasingly rely on regionally-aligned US forces to assist and manage unexpected healthcare emergencies as they develop. Thus, there is an urgen- cy to continually improve joint adaptability, versatility, and training to cope with uncertainty and complexity The Growing Challenges of Vector-Borne Diseases to Regionally-Aligned Forces COL Leon L. Robert, Jr, MS, USA COL Mustapha Debboun, MS, USA Abstract The long-term strategic focus of US foreign policy has pivoted to the Pacific, but tensions in the Middle East require constant attention in the present. As our current role in Afghanistan diminishes, we must seize the opportunity to refocus on the new priority of regionally-aligned forces. The short-term reality requires first re- establishing core warfighting competencies of a smaller Army and then building the capacity of forces focused on regional alignment. The continuing threat of vector-borne and other infectious diseases will present growing challenges to US forces focused on regional alignment and engagement. Greater understanding of these threats, host nation vulnerabilities and capabilities, and the regional presence of international and nongovernmental organizations will enable US forces to respond and engage more effectively and appropriately to accomplish assigned missions and future contingencies. Effective vector surveillance and control has a longstanding and proven record of preventing, reducing, and eliminating vector-borne diseases and must remain a focus of re- gionally-aligned forces. Operational readiness of armed forces continues to rely heavily on vector surveillance and control, and on personal protection strategies. Regionally-aligned forces must also work closely with the US Department of State and US Agency for International Development, international governments, govern- mental and nongovernmental organizations, and private organizations operating in the region and know how to effectively interact with these diverse organizations. In addition, a working knowledge of a host country’s public health policy, capabilities and economic realities will be essential. Teamwork with previously unfamiliar groups and organizations will be an essential component of working in regional environments and can present unfamiliar tasks for traditionally-trained military units.
  • 9. July – September 2014 7 in military preventive medicine and public health as it relates to infectious disease surveillance and control.3 Challenges Ahead United States forces will continue to face challenges in remote lands ranging from regular and irregular wars, humanitarian relief and reconstruction, to sustained global engagement. The challenge to force health pro- tection (FHP) planners and leaders is best summarized by the FHP problem statement below. The problem that faces the joint force is to determine how to more effectively provide health protection to a force that will operate in a complex and diverse opera- tional environment; confront a range of traditional and new adversaries and threats; employ and integrate new technologies; and collaborate with other organizations, agencies, nations and cultures.4(p15) The importance of this problem statement is reinforced by the Director-General of the World Health Organiza- tion who warns that emerging diseases have become a much larger menace in a world characterized by high mobility and unstable economies.5 Also, climate change, widespread land-use change, globalization of trade and travel, and social upheaval are driving the emergence and changing patterns of zoonotic (animal diseases transmissible to humans under natural conditions) and vector-borne diseases (infectious diseases transmitted host-to-host by another animal, usually an arthropod) worldwide. Throughout history, zoonotic diseases and vector-borne diseases have severely reduced the fighting strength of armies and changed the course of military operations. Since World War I, these infectious diseases are no lon- ger the main causes of morbidity and mortality among military personnel. However, even with modern medi- cine and therapeutics, zoonotic and vector-borne infec- tious diseases continue to be an important threat to US forces both in the United States and worldwide.6 Con- tinual progress in modern medicines, hygiene, and vec- tor control has lessened the effects of some vector-borne diseases (eg, plague, yellow fever, and epidemic typhus) while others such as malaria and dengue fever remain a military concern and new potential threats continue to emerge, West Nile encephalitis and chikungunya fever, for example.7 As neither effective medication nor vac- cines are available for some of these diseases, vector control remains pivotal. Therefore, operational readi- ness of armed forces continues to rely heavily on vec- tor surveillance and control, and on personal protec- tion strategies. Without aggressive application of both vector control and personal protection strategies, these diseases may again have the same devastating effect on service member health and military readiness as they did in the past. The Global Challenge of Vector-Borne Diseases The global challenge of infectious diseases cannot be underestimated; nearly half of the world’s population is at risk from at least one type of vector-borne pathogen.8 Vectors like mosquitoes, ticks, and fleas transmit para- sites, viruses, or bacteria between people or between animals and people. Because of the increasing threat of vector-borne diseases, the World Health Organization (WHO) selected vector-borne diseases as the theme of the 2014 World Health Day.8 Numerous publications and workshops have reviewed the relationship between glob- al change and vector-borne diseases and have provided specific recommendations for detection and control ca- pabilities, improving and coordinating surveillance, di- agnosis, and response to disease outbreaks.9-11 Vector-borne diseases account for 17% of the estimated global burden of all infectious diseases.12 Every year more than one billion (109 ) people are infected and more than one million people die from vector-borne diseases, including malaria, dengue, schistosomiasis, leishmani- asis, Chagas disease, yellow fever, lymphatic filariasis, and onchocerciasis.11 Forty percent of the world’s pop- ulation is at risk from dengue virus; there are an esti- mated 390 million dengue infections each year in over 100 countries. Dengue is the world’s fastest growing vector-borne disease, with a 30-fold increase in disease incidence over the last 50 years. Southeast Asia and Latin America are especially affected, but dengue also occurs in Africa, where cases are less often diagnosed. Malaria is a vector-borne disease that is one of the most severe public health problems worldwide. It is a leading cause of death and disease in many developing coun- tries, where young children and pregnant women are the groups most affected. The WHO estimates that in 2012, there were 207 million cases of malaria resulting in 627,000 deaths.11 Global trade, rapid international travel, and environ- mental changes such as climate change and urbaniza- tion are causing vectors and vector-borne diseases to spread beyond borders. At the same time, the world is facing a severe shortage of entomologists and vec- tor control experts.11 Very few African countries have entomology programs at the undergraduate university level, and some countries have few trained entomolo- gists. Many national and local governments, especially in Africa and Asia, are reducing their financial com- mitments to vector-borne disease surveillance, control, and treatment. This has resulted in the silent increase in
  • 10. 8 http://www.cs.amedd.army.mil/amedd_journal.aspx vector-borne disease morbidity and mortality in these areas. These realities will place increased burden on re- gionally-aligned US forces as they engage and reengage with regional allies and threats. More closely related to the present and future challenges to surveillance and control of zoonotic and vector-borne diseases are global threats in sanitation and health, espe- cially the most daunting challenges in megacities.13,14 The number of these megacities with populations in excess of 10 million people is projected to reach 27 by 2015.15 These cities will not only pose significant military and public health threats to deployed forces, but will certainly be characterized by growing lawlessness, increasing pover- ty, and decreaseng essential services. Public health em- phasis to eliminate insect vectors and control associated diseases must grow away from “blanket” application of pesticides to more integrated and sustainable approaches. These integrated approaches must include sound envi- ronmental management practices, community education and participation, mobilizing community resources, and minimal reliance on routine pesticidal spraying.11 DoD Force Health Protection for Regionally-Aligned Forces Department of Defense Joint Force Health Protection (JFHP) Concept of Operations (CONOPS) strategic guidance highlights the need for improving joint warf- ighting through JFHP transformation and incorporating that transformation into the respective military services’ operational level JFHP in support of regionally-aligned forces.16 In 2011, the FHP CONOPS4 was published as part of the overarching Health Readiness CONOPS.17 The FHP CONOPS creates a roadmap for significantly improving Military Health System joint interoperability and mission effectiveness. The FHP CONOPS supports rigorous assessment and analysis of force health pro- tection-related capabilities through analysis of existing and new requirements, capability gaps, and shortfalls. Subsequently, follow-on recommendations will be made for appropriate materiel and nonmateriel solutions to be presented and adjudicated as part of broader DoD joint capabilities. Regionally-aligned forces will increasingly rely on early warning health protection detection systems, including the capability to establish fixed in-theater, early warn- ing of infectious disease trends and characterization that will require the capability of access to Level IV and V diagnostic laboratories. The full and appropriate use of these diagnostic facilities will certainly necessitate advanced training of military preventive medicine per- sonnel so they fully understand the requirements and capabilities of rapid diagnostic labs. Regionally-aligned forces will also require a full under- standing of the capabilities of overseas DoD regional research and surveillance laboratories. There are Na- val Medical Research Units in Phnom Penh, Cambo- dia (NAMRU-2), Cairo, Egypt (NAMRU-3) and Lima, Peru (NAMRU-6). In addition, the Walter Reed Army Institute of Research has Special Foreign Activity labo- ratories in Nairobi, Kenya (US Army Medical Research Unit-Kenya); Sembach, Germany (USAMRU-Europe); and Bangkok, Thailand (US Army Medical Component of the Armed Forces Research Institute of Medical Sci- ences (USAMC-AFRIMS)) with satellite laboratories located in rural Thailand and Nepal. The missions of these laboratories include prevention of psychiatric battle casualties, disease surveillance, vector studies, arboviral transmission, basic research, and vaccine and drug development for enteric diseases (infectious diar- rhea), malaria, tropical viral diseases, and HIV/AIDS to increase the operational readiness of forward-deployed service members. The overseas laboratories continuously conduct disease surveillance as part of the Armed Forces Health Surveil- lance Center’s Global Emerging Infections Surveillance and Response System which performs surveillance for emerging infectious diseases that could affect the US military. This mission is accomplished by orchestrat- ing a global portfolio of surveillance projects, capacity- building efforts, outbreak investigations, and training ex- ercises. Because overseas laboratories interact with host nation medical systems at national and local levels on a frequent (often daily) basis, they have a deep understand- ing of population health trends, needs, and capabilities. Regionally-aligned forces necessarily work closely with the US Department of State, US Agency for Internation- al Development, international governments, nongovern- mental organizations (NGOs), and private organizations operating in the region; so they must understand how to effectively interact with these diverse organizations. In addition, a working knowledge of the host country’s public health policy, capabilities, and economic realities is essential. The provincial reconstruction teams part- nering with the Afghan government to implement the Afghan Ministry of Public Health National Malaria Stra- tegic Plan is an example of implementing the planning considerations for foreign internal defense missions that will also apply in future regionally-aligned missions.3 This knowledge and associated skills are not learned in traditional military operational or training environments. Thus, military and specialty-specific training must in- clude not only traditional military preventive medicine and sanitation topics but be expanded to include country THE GROWING CHALLENGES OF VECTOR-BORNE DISEASES TO REGIONALLY-ALIGNED FORCES
  • 11. July – September 2014 9 THE ARMY MEDICAL DEPARTMENT JOURNAL and region-specific information, to include cultural awareness and rudimentary language skills. This ex- panded skill set will not be learned from existing train- ing courses and programs. New training opportunities must be afforded military preventive medicine person- nel to familiarize them with how to interact with and synergize the efforts of host nation assets, other gov- ernmental agencies, NGOs, and international military partners. This training should start with initial entry training and be a continual process. A Glimpse into the Future? The WHO recently established the integrated vector management (IVM) strategy, a rational decision-making process to optimize use of resources, as an innovative platform for combating vector-borne diseases. The strat- egy is based on the premise that effective control is not the sole responsibility of the health sector but of a wide range of public and private agencies, including local communities. Five key attributions have been identified as being critical to the IVM strategy: (1) advocacy; social mobilization and legislation; (2) collaboration with the health sector and with other sectors; (3) integrated ap- proach; (4) evidenced-based decision making; and (5) ca- pacity building. The ultimate goal is to prevent the trans- mission of vector-borne diseases such as malaria, den- gue, Japanese encephalitis, leishmaniasis, schistosomia- sis, and Chagas disease.18 It has been recently suggested that this new IVM be used for rational decision-making and sustainable vector control in Southern Sudan and an archetype for other similar postconflict environments.19 The signing of the 2005 Comprehensive Peace Agree- ment in South Sudan marked the end of decades of civil war and left the country with enormous infrastructure, human and financial resource constraints, and a weak healthcare system facing a huge burden of several vec- tor-borne diseases.20 The implementation of IVM, more specifically in South Sudan, will require an explicit un- derstanding of spatio-temporal patterns of vector-borne diseases and complicating factors that incorporates and integrates all necessary information, such as geographic information system tools. Establishing a viable IVM strategy will face formidable challenges: environmental, sociocultural, socioeconomic, technical, and program- matic, to name a few. Challenges will be exacerbated by instability, a weak (almost nonexistent) healthcare system, limited access to health services, a paucity of entomological and epidemiological information, and ex- tremely limited skilled personnel to implement the vec- tor control program. The potential for using IVM to integrate successful vec- tor control in South Sudan is enormous. However, the realities of lack of essential physical infrastructure, fi- nancial research and technical expertise, and resources are daunting. Even if successful, the IVM process will be slow and will require a sustained national and in- ternational commitment. A version of this model was used in Afghanistan by provincial reconstruction teams, agribusiness development teams, and civil affairs units to develop the Afghan Ministry of Public Health Na- tional Malaria Strategic Plan, 2008-2013,21 and imple- ment this plan to rebuild the nation-wide Afghanistan malaria control infrastructure.3 This mission required a detailed, advanced knowledge of how other governmen- tal agencies (ie, Department of State and US Agency for International Development), international governments, NGOs, and private organizations were operating in Af- ghanistan, and how to effectively interact with these di- verse organizations. In addition, a working knowledge of the host country’s public health policy, capabilities, and economic realities is essential Military force health protection planners would be well-advised to use the current and future IVM efforts in South Sudan as a case study for future contingency threats to regionally-aligned forces. If successful, IVM may serve as a global strategic framework for preven- tion and control of vector-borne diseases when engaging and assisting regional international and national part- ners and allies. Summary Regionally-aligned US forces will certainly face new and unforeseen challenges, such as emerging and re- emerging vector-borne diseases. These diseases will have increasingly negative effects on human health in developing countries and growing mega-cities. The challenges will require traditional preventive medi- cine training and prevention measures. However, new skills such as increased coordination and cooperation with host nation assets, other governmental agencies, NGOs, and international military partners will be re- quired. These skills must be learned through increased didactic training opportunities and field training expe- riences. Department of Defense and US Army force health protection doctrine and training must continually adapt to these future challenges. References 1. Lopez TC. Future Army forces must be regionally aligned, Odierno says. Army News Service. Octo- ber 24, 2012. Available at: http://www.defense.gov/ news/newsarticle.aspx?id=118316. Accessed May 5, 2014.
  • 12. 10 http://www.cs.amedd.army.mil/amedd_journal.aspx 2. Colacicco-Mayhugh MG, Gosine S, Hughes T, Di- claro J, Larson R, Dunford J. Military entomology in Operation Enduring Freedom, 2010-2011. US Army Med Dep J. July-September 2012:29-35. 3. Robert LL, Rankin SE. The expanding role of military entomologists in stability and counterin- surgency operations. US Army Med Dep J. July- September 2011:12-16. 4. Force Health Protection Concept of Operations (CONOPS). Washington, DC: Office of the As- sistant Secretary of Defense (Health Affairs), US Dept of Defense; November 17, 2011. Available at: http://ommfr.dhhq.health.mil/libraries/conops/ FHP_CONOPS_17_NOV_2011.sflb.ashx. Accessed May 5, 2014. 5. Chan M. Public Health in the 21st Century: Opti- mism in the Midst of Unprecedented Challenges. Speech [Director-General WHO] presented April 3, 2007; Singapore. Available at: http://www.who.int/ dg/speeches/2007/030407_whd2007/en/. Accessed May 5, 2014. 6. Potter R, Mallak C, Gaydos J. Deaths attributed to zoonotic and vector-borne diseases in US military forces, 1998-2009 [poster abstract]. Presented at: Annual Meeting of Infectious Diseases Society of America; October 22, 2011; Boston, MA. Abstract 1269. 7. Pages F, Faulda M. Orlandi-Pradines E, Parola P. The past and present threat of vector-borne dis- eases in deployed troops. Clin Microbiol Infect. 2010;16(3):209-224. 8. World Health Day–vector-borne diseases. Cen- ters for Disease Control and Prevention website. Available at: http://www.cdc.gov/Features/world healthday2014. Accessed May 5, 2014. 9. Sutherst RW. Global change and human vulnera- bility to vector-borne diseases. Clin Microbiol Rev. 2004;17(1):136-173. 10. Institute of Medicine. Vector-borne Diseases: Un- derstanding the Environmental, Human Health, and Ecological Connections. Washington, DC: The National Academies Press; 2008. 11. A Global Brief on Vector-Borne Diseases. Ge- neva, Switzerland: World Health Organiza- tion; 2014. Document number: WHO/DCO/ WHD/2014.1. Available at: http://apps.who.int/ iris/bitstream/10665/111008/1/WHO_DCO_ WHD_2014.1_eng.pdf. Accessed May 5, 2014. 12. Moe C, Rheingans R. Global challenges in water, sanitation and health. J Water Health. 2006;4(suppl 1):41-57. 13. Kahn MMH, Krämer A, Prüfer-Krämer L. Cli- mate change and infectious diseases in megacities of the Indian subcontinent: a literature review. In: Krämer A, Khan MMH, Kraas F, eds. Health in Megacities and Urban Areas. Berlin, Germany; Physica-Verlag:2011:135-152. 14. World Urbanization Prospects: The 2003 Revision. New York, NY: United Nations; 2004. Available at: http://www.un.org/esa/population/publications/ wup2003/WUP2003Report.pdf. Accessed May 5, 2014. 15. Knudsen AB, Slooff R. Vector-borne disease problems in rapid urbanization: new approach- es to vector control. Bull World Health Organ. 1992;70(1):1-6. 16. Joint Force Health Protection Concept of Opera- tions. Washington, DC: US Dept of Defense; July 2007. 17. Health Readiness Concept of Operations (CONOPS). Washington, DC: Office of the As- sistant Secretary of Defense (Health Affairs), US Dept of Defense; January 21, 2010. Available at: http://ommfr.dhhq.health.mil/libraries/conops/ Health_Readiness_CONOPS_21JAN2010.sflb. ashx. Accessed May 5, 2014. 18. World Health Organization. Integrated vector man- agement (IVM) [internet]. Available at: http://www. who.int/neglected_diseases/vector_ecology/ivm_ concept/en/. Accessed May 6, 2014. 19. Chanda E, Govere JM, Macdonald MB, Lako RL, Haque U, Baba SP, Mnzava A. Integrated vector management: a critical strategy for combating vector-borne diseases in South Sudan. Malar J. October 2013;12:369-377. Available at: http://www. malariajournal.com/content/pdf/1475-2875-12-369. pdf. Accessed May 6, 2014. 20. Health Policy Government of Southern Sudan 2007-2011. Juba, Republic of South Sudan: Minis- try of Health; 2006. Available at: http://goss-online. org/magnoliaPublic/en/ministries/Health.html. Accessed May 5, 2014. 21. Draft National Malaria Strategic Plan 2008-2013. Kabul, Afghanistan: Islamic Republic of Afghni- stan Ministry of Public Health; 2007. Available at: http://www.nationalplanningcycles.org/planning- cycle-file-repository/AFG. Accessed May 5, 2014. Authors COL Robert is a Professor and Head, Department of Chemistry and Life Science, US Military Academy, West Point, New York. COL Debboun is Chief, Department of Preventive Health Services, Academy of Health Sciences, US Army Medi- cal Department Center and School, Fort Sam Houston, Texas. He is also the Chairman of the Army Medical De- partment Journal Editorial Review Board. THE GROWING CHALLENGES OF VECTOR-BORNE DISEASES TO REGIONALLY-ALIGNED FORCES
  • 13. July – September 2014 11 Mosquito-borne disease agents can pose a threat to hu- mans, particularly to deployed troops, both in foreign environments and, if imported, domestically. Gaps exist in the fundamental knowledge regarding mosquito vec- tor species, specifically concerning the species complex in subgenera Anopheles, Aedes, and Culex from central Japan. These 3 subgenera include major vector species that are responsible for transmitting malaria, dengue, Japanese B encephalitis, as well as other pathogenic mi- croorganisms in many parts of the world, particularly in Asia. Anopheles Hyrcanus Group consists of several species that are vectors of malaria, filariasis and other mosquito-borne diseases in the Oriental and Palearc- tic regions. Currently, about 30 species have been de- scribed and named.1-3 In 2004, about 27 species were listed in the Hyrcanus Group, with 6 species placed in the Lesteri Subgroup, 4 in the Nigerrimus Subgroup, and 17 in the unassigned subgroup.3 In their 2013 review of the malaria vectors in the Greater Mekong subre- gion, Hii and Rueda4 created the new Sinensis Subgroup that contains those previously unassigned species (An. sinensis Wiedemann, An. pullus Yamada, other 6 spe- cies). Recently, there is more focus on Anopheles Hyr- canus Group in Asia, primarily to clarify the taxonomy of the species complex and to update the distribution records of vectors and related species.4-10 Although sev- eral Anopheles mosquito publications exist, they were not updated to include recent discoveries, taxonomic re- cords, and related pertinent collection data from central Japan.11-14 In 2005, Rueda and others5 noted five species of An. Hyrcanus Group occurring in Japan, namely: An. sinensis, An. engarensis Kanda and Ogama, An. yatsu- shiroensis Miyazaki, An. sineroides Yamada, and An. lesteri Baisas and Hu. In 2013, Imanishi15 recorded for the first time An. belenrae Rueda from Hokkaido, Japan. Anopheles pullus and An. kleini Rueda, the primary ma- laria vectors in South Korea, have never been collected in Japan.16 Known and potential vectors of malaria in the Hyrcanus Group include An. sinensis, An. lesteri, An. belenrae, An. kleini, and An. pullus.16 The purpose of our study was to strengthen mosquito- borne disease biosurveillance capability in Japan by ac- quiring biogeographic vector data from sites identified as having taxonomic and ecological importance, thereby enhancing the knowledge base associated with poten- tial malaria vectors, and incorporating this information as a component of already in-place mosquito surveil- lance programs, including the Walter Reed Biosystemat- ics Unit’s VectorMap/MosquitoMap, and the US Army Public Health Command Regions – North and Pacific mosquito surveillance training programs. Materials and Methods Mosquito Field Collection and Identification Specimen collections were conducted from 2006-2013 from various areas within Kumamoto, Fukuoka, Saga and Nagasaki Prefectures, on Kyushu Island, Japan (Figure 1). Additional specimens were previously col- lected by Dr Motoyoshi Mogi from 1984-2005 from localities in Saga and Nagasaki Prefectures. These pre- fectures were selected because the taxonomic records Mosquito Biosurveillance on Kyushu Island, Japan, with Emphasis on Anopheles Hyrcanus Group and Related Species (Diptera: Culicidae) Leopoldo M. Rueda, PhD Yukiko Higa, PhD Benedict Pagac, BS Kyoko Futami, PhD Masashiro Iwakami, BS Nozomi Imanishi, MS Alexandra R. Spring, MS MAJ Lewis S. Long, MS, USA Maysa T. Motoki, PhD COL Mustapha Debboun, MS, USA James E. Pecor, BS Abstract This report includes the distribution records of the Anopheles (Anopheles) Hyrcanus Group and associated spe- cies in Kyushu Island, Japan, based on our field collections from various localities of 4 prefectures (Fukuoka, Kumamoto, Nagasaki, Saga), primarily from 2002-2013. The status of common and potential mosquito vectors, particularly Anopheles species, in Japan are noted.
  • 14. 12 http://www.cs.amedd.army.mil/amedd_journal.aspx for the Anopheles Hyrcanus Group were unclear or had conflicting information regarding previously reported and described species from this region. The Hyrcanus Group includes all known malaria vector species in Japan5,14 and it is essential to clarify the taxonomy of the group, including geo- graphic distribution records of the group species. The mosquito taxonomic classification used in this paper follows that of Knight and Stone.19 Depending on the habitats (rice paddies, irrigation ditches, permanent and temporary pools, other standing water areas (Figures 2 and 3)), larvae were collected using a standard larval dipper (350 ml, 13 cm diameter) or a white plastic larval tray (25×20×4 cm) (BioQuip, Rancho Dominguez, CA). Each habitat within a location was surveyed for up to one hour or until about 100 larvae were col- lected. The latitude and longitude of each location was recorded using a hand-held global positioning system (GPS) unit (Garmin International, Olathe, KS) set to the WGS84 datum. Sampling locations were photographed using a digital camera to assist in verifying the accuracy of the habitat description. MOSQUITO BIOSURVEILLANCE ON KYUSHU ISLAND, JAPAN, WITH EMPHASIS ON ANOPHELES HYRCANUS GROUP AND RELATED SPECIES (DIPTERA: CULICIDAE) 33.9 33.4 32.9 32.4 31.9 31.4 30.9 128.5 129 129.5 130 130.5 131 131.5 132 Longitude (degrees) Latitude(degrees) Figure 1. Mosquito collection sites on Kyushu Island (right) and Fu- kue Island (left), Japan. Figure 2. Larval habitats of Anopheles (Anopheles) species in the Nagasaki Prefecture, Kyushu Island: (A) rice paddies with ter- races, with closeup of rice seedlings; (B) rice paddy, with closeup of rhizobium rice plants; (C) irrigation ditch; (D) drainage ditch, partially covered by dried grasses (Fukue Island); (E) water well. A B C D E
  • 15. July – September 2014 13 THE ARMY MEDICAL DEPARTMENT JOURNAL Collected larvae were placed in plastic Whirl-Pak bags (118 ml, 8×18 cm) (BioQuip, Rancho Dominguez, CA) and filled approximately ½ full with water from the col- lection site. The Whirl-Pak was then tightly closed to retain air, placed in a cooler, and brought to the labora- tory where the larvae were directly preserved in 100% ethanol for molecular identification. The remaining larvae were individually link-reared to adult stage, as morphological voucher specimens for this work (Figure 4). Emergent adults were pinned on paper points, each given a unique collection number, and identified using diagnostic morphological characters (Figure 5). DNA Isolation and Sequencing For molecular species identification, DNA was isolated from individual larvae, pupae, and adults (1 or 2 legs per adult) by phenol-chloroform extraction, and the PCR amplification protocol, cycling conditions, and direct se- quencing were carried out using standard protocol.17 A fragment of rDNA ITS2 was amplified using the prim- ers 5.8S (5’-ATCACTCGGCTCGTGGATCG-3’) and 28S (5’-ATGCTTAAATTTAGGGGGTAGTC-3’).18 The PCR products were directly sequenced using Big Dye 3.0 (Applied Biosystems, Inc (ABI), Foster, CA) with an ABI 3100 sequencer. Sequences were edited using Sequencher (V4.8, Gene Codes Corporation, Ann Arbor, MI) and aligned in Clustal X.Sequences of An. Hyrca- nus Group species (An.sinensis, An. lesteri) are those of previous studies using the primers therein.17,18 Voucher specimens and collection records will be deposited in the US National Museum of Natural History (US- NMNH) of the Smithsonian Institution, Suitland, MD. Results The summary of collection localities and larval habitats for Anopheles species (primarily An. sinensis and An. lesteri) from 4 prefectures (Fukuoka, Kumamoto, Na- gasaki, Saga) of Kyushu Island, Japan, are presented in the Table (page 18). The map of Kyushu, with collec- tion sites of mosquitoes, is shown in Figure 1. Prior to 2013, larvae of An. sinensis were collected from various habitats either alone or in association with the following Aedes or Culex species: Cx. (Culex) tritaeniorhynchus Giles larvae (in rice fields, irrigation ditches, marsh and drainage areas, ground pits or depressions) in Nagasaki and Kumamoto Prefectures. Aside from An. sinensis, no Anopheles species were collected from any larval habi- tats in association with Aedes or Culex species. In 2013, A B C D E Figure 3. Larval habitats of Anopheles (Anopheles) species on Kyushu Island: (A) stream margin with small pool (arrow) (Kuma- moto Prefecture); (B) stream margin with small water pockets (arrow) (Kumamoto Prefecture); (C) river margin with water pockets (arrow) (Yatsushiro, Kumamoto Prefecture); (D) lotus field (Nagasaki Prefecture); (E) hill or road side ditch with water lilies and grasses (Nagasaki Prefecture).
  • 16. 14 http://www.cs.amedd.army.mil/amedd_journal.aspx MOSQUITO BIOSURVEILLANCE ON KYUSHU ISLAND, JAPAN, WITH EMPHASIS ON ANOPHELES HYRCANUS GROUP AND RELATED SPECIES (DIPTERA: CULICIDAE) A B C D Figure 4. (A) Emergence plastic vials for rearing mosquito larvae and pupae. (B) Newly emerged adult male Anopheles mosquito. (C) Collected Anopheles Hyrcanus Group larvae showing diverse morphology. (D) Anopheles Hyrcanus Group larva, fourth instar, dorsal view. Figure 5. (A) Pinned adult mosquito specimens for deposition in the WRBU, Smith- sonian Institution, National Mosquito Collections. (B) Pinned adult female of Anopheles belenrae, lateral view. A B
  • 17. July – September 2014 15 THE ARMY MEDICAL DEPARTMENT JOURNAL other Hyrcanus Group larvae (still to be identified us- ing molecular sequences) were also found in association with the following: Cx. (Cux.) tritaeniorhynchus, Cx. (Cux.) spp.; Ae. (Finlaya) spp.; Ae. (Ochlerotatus) spp. in rice paddies and irrigation ditches in Nagasaki Prefec- ture (Isahaya, Moriyama, Obama-Unzen, Onakao). During the 2013 survey of various localities in Kyushu Island, the rice paddies where we collected the larvae and pupae of Anopheles Hyrcanus Group had water pH ranging from 6.68-8.61 (mean, 7.77), millivolt- age (175.00-237.00 mV; mean, 215.10) and temperature (30.50°C-32.80°C; mean, 32.10°C). Other water habitats (irrigation ditches, ponds, stream margin, pools, and drainage) that were positive for Anopheles larvae and pupae also exhibited variable pH, mV, and temperatures. Culicine mosquitoes (nonanophelines) collected from Kyushu Island in 2013 included Ae. (Fin.) japonicus (Theobald) from Moriyama and Nagasaki (artificial con- tainers, shrine stone bowls); Ae. (Fin.) togoi (Theobald) from Isahaya (pond); Ae. (Ste.) albopictus (Skuse) from Moriyama and Nagasaki (artificial containers, drainage ditches, shrine stone bowls, tree stumps or holes, tempo- rary seepage); Cx. (Ocu.) bitaeniorhynchus Giles from Moriyama (drainage ditches); and Cx. (Cux.) tritaenio- rhynchus from Moriyama, Obama-Unzen, Hitoyoshi (drainage ditches, irrigation ditches, rice paddies). About 60 mosquito larvae collected from Nagasaki Prefecture could not be identified morphologically, including those in Ae. (Finlaya) from Isahaya; Ae. (Ochlerotatus) from Nomozaki and Onakao; and Cx. (Culex) from Moriyama, Nagasaki, Obama-Unzen, Setoishi, Isahaya, Aikawa, and Onakao. Molecular analysis of those unidentified larval specimens of Aedes and Culex, together with both larvae and adults of An. Hyrcanus Group from 4 prefec- tures, will be completed in the future. Comment Among the Anopheles Hyrcanus Group species, An. pul- lus, An. sinensis, An. lesteri, An. kleini, and An. belenrae are known or potential vectors of vivax malaria in the Korean peninsula and other countries. Anopheles sinen- sis is the most common anopheline species in Japan, in- cluding the Ryukyu Islands.14 It has long been suspected as the most important vector of malaria in Japan, includ- ing Okinawa and Hokkaido. Even though indigenous malaria has disappeared, this vector remains abundant throughout Japan. It is a known vector of malaria in South Korea and China, and it has a wide distribution in Asia.4,5,8-10,14,20 Anopheles lesteri (=anthropophagus) is a very important vector of malaria in China. To clarify and stabilize the taxon, Rueda and others6 designated and described the neotype and alloneotype of An. lesteri. This species was suspected to be an important vector of indigenous malaria in Japan, particularly in Hokkaido where it commonly occurs in great numbers. It is also common in the Ryukyu Islands and has been found more frequently in coastal regions in Honshu and Kyushu.14 Anopheles yatsushiroensis is not known as a vector of in- digenous malaria in Japan. Anopheles belenrae (Figure 4B), first recorded in Japan in 2013 from Hokkaido,15 is a potential vector of vivax malaria in Korea.21 Plasmodium berghei Vincke and Lips, a nonhuman specific parasite, was first detected from An. belenrae adults in South Korea.22 The morphological details of the head, thorax, abdomen, wings, and legs of An. belenrae are shown in the Walter Reed Biosystematics Unit’s website.* The other Hyrcanus Group species (ie, An. sineroides and An. engarensis), as well as several Anopheles (Anopheles) species (An. bengalensis Puri; An. koreicus Yamada and Watanabe; An. lewisi Ludlow; An. lindsayi japonicus Ya- mada; An. omorii Sakakibara; An. saperoi Bohart and Ingram; An. yaeyamaensis Somboon and Harbach), are not known vectors of indigenous malaria in Japan. Most mosquito collections, including Anopheles spe- cies, noted by Tanaka and others14 in 1979, are presently deposited at the National Institute of Infectious Diseas- es (NIID), Tokyo, Japan, where most of the Hyrcanus Group species were examined by author L. M. Rueda during his visit in 2006. In a recent conversation with the authors, Dr Kyoko Sawabe mentioned that there are some possible specimens of An. yatsushiroensis collect- ed by Dr M. Otsuru in 1951 and 1964 on Kyushu Island now deposited at the NIID, Tokyo. These specimens should be examined for further morphological and mo- lecular analysis to clarify the existence of this species. In 2003, Dr Motoyoshi Mogi inquired to check the type specimens of An. yatsushiroensis from the Department of Parasitology (DP), Faculty of Medicine, Kyushu Uni- versity, Fukuoka, Kyushu (reported as the depository of An. yasushiroensis types by Miyazaki12 in 1951). How- ever, Professor Isao Tada (former director of the DP) in- formed Dr Mogi that no type specimens existed at the DP. It may be useful to designate neotypes for An. yatsu- shiroensis, if it is proven as a valid species. Although previous researchers considered An. yatsushi- roensis as a synonym of An. pullus, they used Korean specimens to obtain their molecular and morphological data.23,24 However, because the type locality of An. yat- sushiroensis is in Japan, it is necessary to do a genetic comparison of An. pullus from South Korea with the topotypic specimens from Japan to resolve definitively if the two are synonyms or not. In 1951, Miyazaki,12 who *http://www.wrbu.org/SpeciesPages_ANO/ANO_A-det/ANbln_A- det.html
  • 18. 16 http://www.cs.amedd.army.mil/amedd_journal.aspx first described An. yatsushiroensis, provided elaborate morphological descriptions, ecology, and distributions of this species. We did not collect An. pullus during our previous collections from 2002-2008 in Japan, and no report indicates the existence of An. pullus in that coun- try. Furthermore, An. pullus is considered a major vec- tor of vivax malaria in the Korean peninsula.16 Through biosurveillance, it is also interesting to investigate if an- other major Korean malaria vector, An. kleini, is present in Japan. In our attempt to recollect specimens of the Hyrcanus Group, particularly An. yatsushiroensis, we recently vis- ited numerous localities and conducted extensive larval collections at various habitats in Nagasaki Prefecture and Kumamoto Prefecture (including Yatsushiro City, the type locality of An. yatsushiroensis reported by Mi- yazaki12 ) and neighboring areas from 2006 to 2013. Un- fortunately, we were not able to collect samples of An. yatsushiroensis from 2006 to 2012. Although Dr Sawabe mentioned that there are some pos- sible specimens of An. yatsushiroensis deposited at the NIID, Tokyo, we have not examined them yet. Further- more, more than 200 larvae and adults of An. Hyrcanus Group collected in 2013 from Kumamoto and Nagasaki Prefectures are still being examined and analyzed by morphological and molecular techniques. Molecular data (PCR, sequences) will be reported later, particu- larly from the 2013 specimens for possible presence of An. yatsushiroensis and other species in An. Hyrcanus Group on Kyushu Island. Acknowledgement This research was performed under a Memorandum of Understanding between the Walter Reed Army Insti- tute of Research and the Smithsonian Institution, with institutional support provided by both organizations. We express our sincere appreciation to the follow- ing: Dr Motoyoshi Mogi for arranging the visits of Dr Rueda to Saga and Fukuoka Prefectures, his help in mosquito collections, and for sharing his mosquito specimens; CPT Robert Moore and SGT J. Santano for their help in collecting mosquito samples from Ku- mamoto Prefecture; Professor Y. Oneda, for his help in collecting samples and guiding us in locating larval habitats in Akagawa and Takegima, Fukuoka Prefec- ture and Tosu City, Saga Prefecture. Special thanks go to Dr Noburo Minakawa, particularly for making the arrangements for our visit to Nagasaki, and Dr Kyoko Sawabe for correspondence and invitation to visit and examine the mosquito collections at NIID, Tokyo. References 1. Rueda LM. Two new species of Anopheles (Anoph- eles) Hyrcanus Group (Diptera: Culicidae) from the Republic of South Korea. Zootaxa. 2005;941:1-26. 2. Ramsdale CD. Internal taxonomy of the Hyrcanus Group of Anopheles (Diptera: Culicidae) and its bearing on the incrimination of vectors of malar- ia in the west of the Palearctic Region. European Mosq Bull. 2001;10:1-8. 3. Harbach RE. The classification of genus Anoph- eles (Diptera: Culicidae): a working hypothesis of phylogenetic relationships. Bull Entomol Res. 2004;94:537-553. 4. Hii J, Rueda LM. Malaria vectors in the Greater Mekong Subregion: overview of malaria vec- tors and remaining challenges. Southeast Asian J Trop Med Public Health. 2013;44(suppl 1):73-165, 306-307. 5. Rueda LM, Iwakama M, O’Guinn M, Mogi M, Prendergast BF, Miyagi I, Toma T, Pecor JE, Wilk- erson RC. Habitats and distribution of Anopheles sinensis and associated Hyrcanus Group in Japan. J Am Mosq Control Assoc. 2005;21(4):458-463. 6. Rueda LM, Wilkerson RC, Li C. Anopheles (Anopheles) lesteri Baisas and Hu (Diptera: Cu- licidae): neotype designation and description. Proc Entomol Soc Washington. 2005;107(3):604-622. 7. Rueda LM, Ma Y, Song GH, Gao Q. Notes on the distribution of Anopheles (Anopheles) sinensis Wi- edemann (Diptera: Culicidae) in China and the sta- tus of some Anopheles Hyrcanus Group type speci- mens from China. Proc Entomol Soc Washington. 2005;107(1):235-238. 8. Rueda LM, Kim HC, Klein TA, Pecor JE, Li C, Sithiprasasna R, Debboun M, Wilkerson RC. Distribution and larval habitat characteristics of Anopheles Hyrcanus Group and related mosquito species (Diptera: Culicidae) in South Korea. J Vec- tor Ecol. 2006;31(1):199-206. 9. Rueda LM, Zhao T, Ma YJ, Gao Q, Guo Ding Z, Khuntirat B, Sattabongkot J, Wilkerson RC. Up- dated distribution records of the Anopheles (Anoph- eles) hyrcanus species-group (Diptera: Culicidae) in China. Zootaxa. 2007;1407:43-55. 10. Rueda LM, Gao Q. New records of Anopheles belenrae Rueda (Diptera: Culicidae) in North Ko- rea. Proc Entomol Soc Wash. 2008;110:523-524. 11. Miyake M. Study on the anopheline mosquitoes in Kyushu. Hukuoka Acta Med. 1950;41:918-927. 12. Miyazaki I. On a new anopheline mosquito Anoph- eles yatsushiroensis n. sp. found in Kyushu, with some remarks on two related species of the genus. Kyushu Mem Med Sci. 1951;2:195-206. MOSQUITO BIOSURVEILLANCE ON KYUSHU ISLAND, JAPAN, WITH EMPHASIS ON ANOPHELES HYRCANUS GROUP AND RELATED SPECIES (DIPTERA: CULICIDAE)
  • 19. July – September 2014 17 THE ARMY MEDICAL DEPARTMENT JOURNAL 13. Otsuru M, Ohmori Y. Malaria studies in Japan af- ter World War II. Part II. The search for Anopheles sinensis sibling species group. Japan J Exp Med. 1960;30:33-65. 14. Tanaka K, Mizusawa K, Saugstad ES. A revision of the adult and larval mosquitoes of Japan (includ- ing the Ryukyu Archipelago and the Ogasawara Islands) and Korea (Diptera: Culicidae). In: Con- tributions of the American Entomological Institute. Vol 16. Gainesville, Florida: American Entomolog- ical Institute; 1979:1-987. 15. Imanishi N. Morphological and Phylogenetic Study of Anopheles belenrae First Recorded from Hokkaido, Japan [master’s thesis]. Kanagawa, Ja- pan: Meiji University; 2013. 16. Klein TA, Kim HC, Lee WJ, Rueda LM, et al. Re- emergence, persistence and surveillance of vivax malaria and its vectors in the Republic of Korea. In: Robinson WK, Bajoni D, eds, Proceedings of the Sixth International Conference on Urban Pests, Budapest, Hungary. 2008. 325-331. Available at: http://www.icup.org.uk/reports/ICUP892.pdf. Ac- cessed May 7, 2014. 17. Wilkerson RC, Li C, Rueda LM, Kim HC, Klein TA, Song GH, Strickman D. Molecular confirma- tion of Anopheles (Anopheles) lesteri from the Re- public of South Korea and its genetic identity with An. (Ano.) anthropophagus from China (Diptera: Culicidae). Zootaxa. 2003;378:1-14. 18. Li C, Lee JS, Groebner JL, Kim HC, Klein TA, O’Guinn ML, Wilkerson RC. A newly recognized species in the Anopheles Hyrcanus Group and mo- lecular identification of related species from the Republic of South Korea (Diptera: Culicidae). Zoo- taxa. 2005;939:1-8. 19. Knight K, Stone A. A Catalog of the Mosquitoes of the World (Diptera: Culicidae). Vol 6. College Park, Maryland: Entomological Society of Amer- ica; 1977. 20. Harrison BA, Scanlon JE. Medical entomology studies – II. The subgenus Anopheles in Thai- land (Diptera: Culicidae). In: Contributions of the American Entomological Institute. Vol 12, No. 1. Gainesville, Florida: American Entomological In- stitute; 1979:1-307. 21. Rueda LM, Li C, Kim HC, Klein TA, Foley DH, Wilkerson RC. Anopheles belenrae, a potential vector of Plasmodium vivax in the Republic of Ko- rea. J Am Mosq Control Assoc. 2010;26(4):430-432. 22. Harrison GF, Foley DH, Rueda LM, et al. Plasmo- dium-specific molecular assays produce uninter- pretable results and non-Plasmodium spp. sequenc- es in field-collected Anopheles vectors. Am J Trop Med Hyg. 2013;89(6):1117-1121. 23. Hwang UW, Yong TS, Ree HI. Molecular evi- dence for synonymy of Anopheles yatsushiroen- sis and An. pullus. J Am Mosq Control Assoc. 2004;20(2):99-104. 24. Shin EH. Hong HK. A new synonym of Anopheles (Anopheles) pullus Yamada, 1937: A. (A.) yatsushi- roensis Miyazaki, 1951. Kor J Entomol. 2001;31:1-5. Authors Dr Rueda is a Research Entomologist, Principal Investi- gator, and Acting Chief of the Walter Reed Biosystemat- ics Unit, Entomology Branch, Walter Reed Army Insti- tute of Research, located at the Smithsonian Institution, Museum Support Center, Suitland, Maryland. Mr Benedict Pagac is the Chief, Entomology Section at the US Army Public Health Command Region-North, Fort George G. Meade, Maryland. Mr Iwakami is an Entomologist at the US Army Public Health Command-Pacific, Entomology Program. Camp Zama, Japan. Ms Spring is a Molecular Biologist at the Entomology Section, US Army Public Health Command Region- North, Fort George G. Meade, Maryland. Dr Motoki is a Postdoctoral Entomologist at the Ento- mology Department, Smithsonian Institution, Museum Support Center, Suitland, Maryland. Mr Pecor is a Museum Specialist at the Walter Reed Biosystematics Unit, Entomology Branch, Walter Reed Army Institute of Research, located at the Smithsonian Institution, Museum Support Center, Suitland, Maryland. Dr Higa, Dr Futami, and Ms Imanishi are Assistant Pro- fessors and Graduate Student, respectively, at the De- partment of Vector Ecology and Environment, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki City, Nagasaki, Japan. MAJ Long is currently on training leave from his po- sition as Chief of the Walter Reed Biosystematics Unit, Entomology Branch, Walter Reed Army Institute of Re- search, located at the Smithsonian Institution, Museum Support Center, Suitland, Maryland. COL Debboun is the Chief of the Department of Preven- tive Health Services, Academy of Health Sciences, US Army Medical Department Center & School, Fort Sam Houston, Texas. He is also the Chairman of the Army Medical Department Journal Editorial Review Board.
  • 20. 18 http://www.cs.amedd.army.mil/amedd_journal.aspx MOSQUITO BIOSURVEILLANCE ON KYUSHU ISLAND, JAPAN, WITH EMPHASIS ON ANOPHELES HYRCANUS GROUP AND RELATED SPECIES (DIPTERA: CULICIDAE) SummaryofcollectionlocalitiesandlarvalhabitatsforAnopheles(Anophleles)in4prefecturesofKyushuIsland,Japan(part1of3). PrefectureLocationGridcoordinatesCollectiondateStageCollectorHabitat typeb Collection No. Anopheles (Anopheles)Species FukuokaAkagawa,OgoriCity33.34975N/130.51352E19-20Sep2008AdultaL.M.Rueda,Y.OnedaRCJP08-9sinensis Fukuoka Takejima,Yasutake- mache,Kurume 33.34975N/130.54597E19-20Sep2008AdultaL.M.Rueda,Y.OnedaRCKP08-10sinensis KumamotoAmitsu,UtoCity32.69973N/130.60432E23Sep2008AdultaL.M.Rueda,M.Iwakami, J.Santano ID,RPJP08-17,18sinensis Kumamoto GyokutoCity, TamanaCounty 32.91638N/130.62565E22Sep2008AdultaL.M.Rueda,M.Iwakami, J.Santano POJP08-14sinensis KumamotoHitoyoshi32.22652N/130.77038E12Jul2013Larva L.M.Rueda,B.Pagac, M.Iwakami ID,RH, RP JP13-19,21HyrcanusGroupc Kumamoto Katoshrine, Yatsushiro 32.47998N/130.57202E15Sep2008AdultaL.M.Rueda,M.Iwakami, J.Santano DDJP08-2lesteri KumamotoLakeEzu32.77755N/130.73855E24Sep2008Adult L.M.Rueda,M.Iwakami, J.Santano LMJP08-19sinensis KumamotoMatsubase,UkiCity32.65355N/130.67050E30Aug2006AdultaM.Iwakami,R.MooreRPJP06-2-37A,40Alesteri KumamotoMatsubase,UkiCity32.65625N/130.66788E17Sep2008AdultaL.M.Rueda,M.Iwakami, J.Santano RPJP08-7,8lesteri KumamotoMatsubase,UkiCity32.65355N/130.67050E30Aug2006AdultaM.Iwakami,R.MooreRP JP06-2-1A,2A,3A, 4A,6A,25A; JP08-5,6 sinensis KumamotoSumiyoshi,UtoCity32.70005N/130.60015E22Sep2008AdultaL.M.Rueda,M.Iwakami, J.Santano IDJP08-17Bsinensis KumamotoTakasima,Yatsushiro32.52158N/130.57750E15Sep2008AdultaL.M.Rueda,M.Iwakami, J.Santano HD,WTJP08-1,4lesteri Kumamoto TamanaCity, TamanaCounty 32.91638N/130.54523E22Sep2008AdultaL.M.Rueda,M.Iwakami, J.Santano RPJP08-15,16sinensis KumamotoUekiCity32.88017N/130.68148E22Sep2008AdultaL.M.Rueda,M.Iwakami, J.Santano ID,RPJP08-12,13lesteri KumamotoUto32.69660N/130.66845E29Aug2006AdultaM.Iwakami,R.MooreRPJP06-1-50A,51Alesteri KumamotoUto32.69660N/130.66845E29Aug2006AdultaM.Iwakami,R.MooreRP JP06-1-1A,2A,3A, 4A,5A,7A,16A sinensis KumamotoYatsushiro32.50033N/130.61932E11Jul2013 Larva,pupa, adulta L.M.Rueda,B.Pagac, M.Iwakami SPJP13-18HyrcanusGroupc KumamotoYatsushiro32.52158N/130.57750E16Sep2008AdultaL.M.Rueda,M.Iwakami, J.Santano RPJP08-3sinensis Nagasaki Ariake-cho, Nagasaki 32.69414N/130.32505E20Jul1988AdultaRCJPM-5sinensis NagasakiGoto,FukueIsland32.67867N/128.76802E17Jul2013Larva,pupa L.M.Rueda,B.Pagac, M.Iwakami IDJP13-31HyrcanusGroupc NagasakiGoto,FukueIsland32.67867N/128.76802E17Jul2013LarvaL.M.Rueda,B.Pagac, M.Iwakami RPJP13-30HyrcanusGroupc aFieldcollectedlarvaeorpupae,rearedtoemergedadults. bDD,drainageditch;HD,hillorroadsideditch;ID,irrigationditch;LM,lakemargin;PO,pond;RC,restingatcowshedorcattlebarn;RH,rockhole,pool;RP,ricepaddy;SP,streamorrivermarginor pool;WT,watertank,troughorPVCtubewaterer. cDNAisolationandsequencingstilltobecompleted.
  • 21. July – September 2014 19 THE ARMY MEDICAL DEPARTMENT JOURNAL SummaryofcollectionlocalitiesandlarvalhabitatsforAnopheles(Anophleles)in4prefecturesofKyushuIsland,Japan(part2of3). PrefectureLocationGridcoordinatesCollectiondateStageCollectorb Habitat typec Collection No. Anopheles (Anopheles)Species NagasakiGoto,FukueIsland32.67867N/128.76802E17Jul2013AdultaL.M.Rueda,B.Pagac, M.Iwakami RPJP13-30HyrcanusGroupd NagasakiGoto,FukueIsland32.67867N/128.76802E17Jul2013Larva L.M.Rueda,B.Pagac, M.Iwakami RPJP13-32HyrcanusGroupd NagasakiGoto,FukueIsland32.67867N/128.76802E17Jul2013AdultaL.M.Rueda,B.Pagac, M.Iwakami RPJP13-32HyrcanusGroupd NagasakiIsahaya32.81308N/130.12767E12Jul2013AdultaNG1POJP13-23HyrcanusGroupd NagasakiIsahaya32.81308N/130.12767E12Jul2013LarvaNG1POJP13-23HyrcanusGroupd NagasakiIsahaya32.81308N/130.12767E12Jul2013LarvaNG1RPJP13-25HyrcanusGroupd Nagasaki Isahaya-shi, Kamiimuta, Moriyama-cho 32.80759N/130.10737E27May2006AdultT.Yoshio-JPM-5sinensis NagasakiMikawa-machi32.78590N/129.88779E27May1962AdultNU-JPM-5sinensis NagasakiMoriyama32.83508N/130.11372E9Jul2013AdultaL.M.Rueda,B.Pagac, M.Iwakami RPJP13-7HyrcanusGroupd NagasakiMoriyama32.83508N/130.11372E9Jul2013Larva L.M.Rueda,B.Pagac, M.Iwakami RPJP13-8HyrcanusGroupd NagasakiNagasaki32.77217N/129.86950E20Jun1989AdultM.MogiRPJPM-5sinensis NagasakiNomozaki32.58570N/129.75660E15Jul2013LarvaNG2ACJP13-26HyrcanusGroupd NagasakiObama-Unzen32.71354N/130.20073E10Jul2013Larva L.M.Rueda,B.Pagac, M.Iwakami RPJP13-14HyrcanusGroupd NagasakiObama-Unzen32.71354N/130.20073E10Jul2013Larva L.M.Rueda,B.Pagac, M.Iwakami RPJP13-15HyrcanusGroupd NagasakiObama-Unzen32.71354N/130.20073E10Jul2013Larva L.M.Rueda,B.Pagac, M.Iwakami WCJP13-16HyrcanusGroupd NagasakiObama-Unzen32.71354N/130.20073E10Jul2013AdultaL.M.Rueda,B.Pagac, M.Iwakami WCJP13-16HyrcanusGroupd NagasakiOnako32.88408N/129.69598E16Jul2013LarvaNG2RPJP13-29HyrcanusGroupd NagasakiTogitsu32.82683N/129.84866E 7-9Aug1956; 22Jul1962 AdultNU-JPM-5sinensis NagasakiTsushima34.17745N/129.29039E27May1962AdultNU-JPM-5sinensis NagasakiUtzutzugawa32.79328N/129.92803E9Jul2013Larva L.M.Rueda,B.Pagac, M.Iwakami RPJP13-13lindsayijaponicus NagasakiUtzutzugawa32.79328N/129.92803E9Jul2013Larva L.M.Rueda,B.Pagac, M.Iwakami RPJP13-13HyrcanusGroupd SagaFukutomi33.17567N/130.17284E 13,20,28Aug 2005;11,13, 24Sep2005; 10Oct2005; AdultM.MogiLFJPM-22-1sinensis aFieldcollectedlarvaeorpupae,rearedtoemergedadults. bNG1indicatesL.M.Rueda,B.Pagac,M.Iwakami,Y.Higa,K.Futami,N.Imanishi.NUindicatesNagasakiUniversity,EntomologyCollection.NG2indicatesL.M.Rueda,B.Pagac,M.Iwakami, Y.Higa,K.Futami. cAC,artificialcontainers(tires,plasticjugs,kettle,etc);CA,roadditchorsmallcanal;ID,irrigationditch;LF,lotusfield;PO,pond;RP,ricepaddy;WC,waterwell/cistern. dDNAisolationandsequencingstilltobecompleted.
  • 22. 20 http://www.cs.amedd.army.mil/amedd_journal.aspx MOSQUITO BIOSURVEILLANCE ON KYUSHU ISLAND, JAPAN, WITH EMPHASIS ON ANOPHELES HYRCANUS GROUP AND RELATED SPECIES (DIPTERA: CULICIDAE) Summary of collection localities and larval habitats for Anopheles (Anophleles) in 4 prefectures of Kyushu Island, Japan (part 3 of 3). Prefecture Location Grid Coordinates Collection date Stage Collector Habitat typeb Collection No. Anopheles (Anopheles) Species Saga Kase 33.23807N 130.25824E 17 Sep 2005 Adult M. Mogi ID JPM-21-1, -2, -3 sinensis Saga Kinyu 33.24204N 130.29149E 2, 5, 10 Jun 1986 Adult M. Mogi RP JPM-8, 9 lesteri Saga Morita 33.09454N 130.10894E 16 May 1995 Adult M. Mogi CA JPM-14 sinensis Saga Nabeshima 33.27991N 130.26614E 30 May 1985; 14 Jun 1985; 3, 5, 6, 7, 20 Jun 1986; 9 Jun 1990; 7 Jun 1995; 3 Jun 1996 Adult M. Mogi LF JPM-4, 5, 6, 7 lesteri Saga Shiroishi 33.17837N 130.14394E 7 May 2000 Adult M. Mogi RP JPM-19-1 lesteri Saga Shiroishi 33.17837N 130.14394E 3 Nov 1997; 7 May 2000 Adult M. Mogi NE JPM-14, 15, 19-2, 19-3, 19-5, 19-5 sinensis Saga Yamato-cho 33.14766N 130.14832E 10 Apr 2000 Adult T. Sunahara CA JPM-18 lesteri Saga Yamato-cho 33.14766N 130.14832E 5 Jun 1986; 24, 25, 26 Apr 2000; 10, 11, 12, 25 May 2000; 24, 25 Apr 2004 Adult T. Sunahara, M. Mogi CA JPM-8, 15, 16, 17, 18 sinensis Saga Tosu City 33.34463N 130.51352E 20 Sep 2008 Adulta L. M. Rueda, Y. Oneda GT JP08-11 lesteri aField collected larvae or pupae, reared to emerged adults. bCA, road ditch or small canal; GT, ground pit or depression; ID, irrigation ditch; LF, lotus field; NE, caught by insect net; RP, rice paddy. Articles published in the Army Medical Department Journal are indexed in MEDLINE, the National Library of Medicine’s (NLM’s) bibliographic database of life sciences and biomedical information. Inclusion in the MEDLINE database ensures that citations to AMEDD Journal content will be identified to researchers during searches for relevant information using any of several bibliographic search tools, including the NLM’s PubMed service.
  • 23. July – September 2014 21 Vector-borne diseases remain a significant cause of dis- ease for US service members throughout the US Euro- pean Command, Africa Command, and Central Com- mand, thereby requiring strong surveillance systems and outbreak responses.1 There is a need for surveil- lance or reference laboratories to perform standardized, high-throughput testing capable of meeting the needs of a changing military environment and response efforts. An understanding of the significance and location of pathogens is important in the development of prevention programs for human and animals, as well as pest-control activities. Although there are numerous tests to detect arthropod-borne diseases, polymerase chain reaction (PCR) assays offer both high sensitivity and specificity for either DNA or RNA recovered from field-collected arthropod samples.2-4 The use of real-time PCR assays offers not only the option of high-throughput testing in an otherwise time and labor intensive procedure, but also removes the need for any postamplification pro- cessing (ie, agarose gel electrophoresis). This study describes the development, validation, and standard- ization of real-time PCR and reverse transcription real- time PCR assays to detect Anaplasma phagocytophilum, Ehrlichia spp, Borrelia spp (B burgdorferi, B afzelii, and B garinii), Crimean-Congo hemorrhagic fever vi- rus, Leishmania, sandfly fever Sicilian virus, dengue vi- rus, Plasmodium spp, and chikungunya virus at the US Army Public Health Command Region-Europe. Materials and Methods At the time of this writing, all methods discussed in the article are accredited by the American Association for Laboratory Accreditation and placed on Laboratory Sci- ences’ ISO/IEC 17025:2005 Biological Scope of Accred- itation (certificate number 2138.01). USAPHCR-Europe LS is registered by Bureau Veritas under the DIN EN ISO 9001:2008 Quality Management System standard (certificate number DE002197-1) and the DIN EN ISO 14001:2009 Environmental Management System stan- dard (certificate number DE002198-1). Homogenization of Arthropods Negative mosquitoes, sandflies (obtained from the Wal- ter Reed Army Institute of Research, Silver Spring, MD), and larvae, nymph, and adult ticks (Bayer Health- Care AG: Animal Health, Germany) were stored in High-Throughput Vector-Borne Disease Environmental Surveillance By Polymerase Chain Reaction According To International Accreditation Requirements Marty K. Soehnlen, PhD, MPH* SPC Carlos J. Gomez, USA CPT Stephen L. Crimmins, MS, USA* Michael E. Cross, MS Andrew S. Clugston, MS Charles N. Statham, PhD Nina Gruhn, MS Abstract Although vector-borne diseases are specific to the region of the host, there is a necessity for surveillance or reference laboratories to perform standardized, high-throughput testing capable of meeting the needs of a changing military environment and response efforts. The development of standardized, high-throughput, semiquantitative real-time and reverse transcription real-time polymerase chain reaction (PCR) methods allows for the timely dissemination of data to interested parties while providing a platform in which long-term sample storage is possible for the testing of new pathogens of interest using a historical perspective. PCR testing allows for the analysis of multiple pathogens from the same sample, thus reducing the workload of entomologists in the field and increasing the ability to deter- mine if a pathogen has spread beyond traditionally defined locations. US Army Public Health Command Region- Europe (USAPHCR-Europe) Laboratory Sciences (LS) has standardized tests for 9 pathogens at multiple life stages. All tests are currently under international accreditation standards. Using these PCR methods and laboratory model, which have universal Department of Defense application, the USAPHCR-Europe LS will generate quality data that is scientifically sound and legally defensible to support force health protection for the US military in both deployed and garrison environments. *Dr Soehnlen and CPT Crimmins contributed equally to first authorship of this article.
  • 24. 22 http://www.cs.amedd.army.mil/amedd_journal.aspx 70% ethanol at -80°C. Samples were transferred to 1.5 mL round-bottom tubes (DNA LoBind, Eppendorf AG, Hamburg, Germany) with 400 µL phosphate buff- ered saline (Sigma-Aldrich, St. Louis, MO). A total of 5 mosquitoes or sandflies were added to tubes and crushed with a micropestle (Kimble Chase, NJ). One tick, regardless of life-stage, was added to each tube and crushed with a micropestle. One 5-mm tungsten carbide bead (Qiagen, Netherlands) was added to each tube. Samples were homogenized using the TissueLyser system (Qiagen, Netherlands) for 10 minutes at 20 Hz. After a short centrifugation step of 20 to 60 seconds at 9,000 rpm, 200 µL of the supernatants were collected and transferred to sample processing cartridges or new 1.5 mL round-bottom tubes. Automated Nucleic Acid Extraction Automated nucleic acid extraction was performed using the MagNA Pure96 (Roche Applied Sciences, Germa- ny) and the MagNA Pure LC (Roche Applied Sciences, Germany). A total of 200 µL of arthropod homogenate supernatant was added to 300 µL of external lysis buf- fer for the MagNA Pure LC using MagNA Pure LC small volume total nucleic acid kit with the External Lysis Protocol (Roche Applied Sciences, Germany) ac- cording to manufacturer instructions. A total of 200 µL arthropod homogenate supernatant was extracted using MagNA Pure96 DNA and viral NA small volume kit with the Universal Pathogen Protocol. A total volume of 50 µL nucleic acid was acquired from samples on both automated extraction instruments. Isolated total nucleic acids were stored at -20°C or below until PCR analysis was performed. DNA and RNA Absorbance Analysis The 260/280 ratio, 230 nm, and 300 nm results for 2 µL extracted sample were analyzed using a NanoQuant plate (Tecan Group Ltd, Switzerland) on the Mx Pro 200 microplate reader (Tecan Group Ltd, Switzerland). Real-Time PCR Real-time PCR reactions were performed using genesig Advanced kit (Primerdesign Ltd, UK) as appropriate for the pathogen being tested and 2X One Step qPCR Master Mix (Primerdesign Ltd, UK). The real-time PCR conditions were: 5 µL of total nucleic acid,10 µL of 2X OneStep qPCR Master Mix, one µL of target primer, one µL of internal positive control primer, and DNAse/ RNAse free water (Qiagen, Netherlands) to adjust the volume to 15 µL. Amplification was performed in Light- Cycler 480 96-well PCR plates (Roche Applied Scienc- es, Germany). Plates were sealed with LightCycler 480 sealing film (Roche Applied Sciences, Germany). The cycling conditions for amplification were performed on a LightCycler 480 (Roche Applied Sciences, Germany) as follows: initial enzyme activation step at 95°C for 10 minutes, and 40 cycles of 95°C for 10 seconds, 65°C for one minute. Melting curve data was collected immedi- ately after cycle 40; one cycle at 60°C for one minute, and 95°C (0.06°C/second) for 10 minutes. Real-Time Reverse Transcription PCR Real-time reverse transcription PCR reactions were performed using genesig Advanced kit and 2X One Step qRT-PCR Master Mix. The real-time reverse tran- scription PCR conditions were as follows: 5 µL of total nucleic acid, 10 µL of 2X One Step qRT-PCR Master Mix, one µL of target primer, one µL of internal positive control primer, and DNAse/RNAse free water to adjust the volume to 15 µL. Amplification was performed in LightCycler 480 96-well PCR plates. Plates were sealed with LightCycler 480 sealing film. The cycling condi- tions for amplification were performed on a LightCy- cler 480 as follows: reverse transcription at 55°C for 10 minutes, enzyme activation step at 95°C for 8 minutes, and 40 cycles of 95°C for 10 seconds, 65°C for one min- ute. Melting curve data was collected immediately after cycle 40; one cycle at 60°C for one minute, and 95°C (0.06°C/second) for 10 minutes. It should be noted that new Master Mix formulas (Precision and Precision- PLUS) were recently introduced by PrimerDesign and have not yet been used by the laboratory. The new con- sumables will be subjected to the same verification and validation procedures presented in this article and in ac- cordance with the LS Quality Management System prior to being incorporated into regular use. Calculations Method detection limits, defined as the minimum con- centration that can be measured and reported with 99% confidence that the value determined is above zero, were determined as the Student’s t test value at the 99% confidence level multiplied by the standard deviation of the data set. Results It was determined that the method detection limit (MDL) for the pathogen kits using 5 adult female arthropods as the matrix for Plasmodium ssp, dengue fever virus, chikungunya virus, Leishmania ssp, and sandfly fever Sicilian virus were 97; 2,500; 25,000; 19,000; and 1,300 copies, respectively. Tick MDLs were determined using one tick per each adult, nymph, and larvae life stage. Anaplasma phagocytophilum adult, nymph, and larvae MDLs were determined to be 6,300; 6,900; and 5,400 copies, respectively. Ehrlichia spp adult, nymph, and larvae MDLs were determined to be 130,000; 240; and 480 copies, respectively. Borrelia spp adult, nymph, and HIGH-THROUGHPUT VECTOR-BORNE DISEASE ENVIRONMENTAL SURVEILLANCE BY POLYMERASE CHAIN REACTION ACCORDING TO INTERNATIONAL ACCREDITATION REQUIREMENTS

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