SHORT COMMUNICATION
Evaluation of Anopheline Mosquitoes (Diptera: Culicidae) from the Republic of Korea for Plasmodium vivax Circumsporozoite Protein RUSSELL E. COLEMAN,1 CHUKREE KIATTIBUT, JETSUMON SATTABONGKOT, JEFFREY RYAN,2 DOUGLAS A. BURKETT,3 HUNG C. KIM,4 WON J. LEE,5 AND TERRY A. KLEIN6 Department of Entomology, Armed Forces Research Institute of Medical Sciences, Bangkok, 10400, Thailand
J. Med. Entomol. 39(1): 244Ð247 (2002)
ABSTRACT As part of an on-going malaria surveillance effort conducted by the U.S. Forces Korea, Republic of Korea (ROK), a total of 28,286 anopheline mosquitoes was tested for the presence of Plasmodium vivax circumsporozoite (CS) protein. Mosquitoes were collected (using a variety of light and baited traps) from 29 locations throughout the ROK (the majority were collected near the de-militarized zone), identiÞed to species, and tested by enzyme-linked immunosorbent assay for the presence of P. vivax 210 and P. vivax 247 CS protein. Recent evidence suggests that characters used to separate Anopheles sinensis Wiedemann from An. lesteri Baisas & Hu are unreliable; therefore, the data have been analyzed by grouping these two species. A total of 25,365 Anopheles sinensis/lesteri, 2,890 An. yatsushiroensis Miyazaki, and 31 An. sineroides Yamada was tested. Of these, one pool of 10 An. sinensis/lesteri collected on 9 September 1999 at Camp Howze and one pool of nine An. sinensis/ lesteri collected on 13 September 1999 at Camp Bonifas were positive for P. vivax 247. KEY WORDS Anopheles, Plasmodium vivax, vivax malaria, diagnostics, enzyme-linked immunosorbent assay, Republic of Korea
MALARIA REEMERGED IN the Republic of Korea (ROK) in 1993 (Chai et al. 1994), followed by two more cases in 1994 (Cho et al. 1994). Since then, the number of new cases has risen exponentially with a total of 6,249 cases by the end of 1998 (Chai 1999). Before 1999, most cases were reported in military personnel in the northwestern part of the country, northern Kyonggi Province and northwestern Kangwon Province (Strickman et al. 2000). Although the proportion of reported malaria has been greater in the civilian population since 1998, many of the civilian cases are reported from military conscripts who have completed their 2-yr obligations. It has been postulated that most of the initial malaria cases in the ROK resulted from bites of sporozoite-infected Anopheles sinensis Wiedmann that were infected in North Korea and subsequently dispersed across the de-militarized zone (DMZ) into the ROK (Chai 1999, Kho et al. 1999, Ree, 2000). However, vivax malaria is now Þrmly established and endemic within the ROK (Lee et al. 1998). 1
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[email protected]. Department of Entomology, Walter Reed Army Institute of Research, Silver Spring, MD 20910-7500. 3 Detachment 3, U.S. Air Force Institute for Environment, Safety, and Occupational Risk Analysis (AFIERA), Okinawa Japan, APO AP 96368. 4 Fifth Medical Detachment, 168th Medical Battalion (Area Support), 18th Medical Command, Unit 14247, APO AP 96205Ð 0020. 5 Korean National Institute of Health, Department of Viral Diseases, 5 Nokbun-Dong, Eunpyung-Gu, Seoul 122Ð701, Republic of Korea. 6 Preventive Services Directorate, 18th Medical Command, Unit 15281, APO AP 96205Ð 0054. 2
Surveillance for vector anopheline mosquitoes associated with the reemergence of vivax malaria in the ROK has primarily focused on determining the seasonality and distribution of presumptive vectors (Kim et al. 1997, 1999; Shim et al. 1997, Strickman et al. 1999, 2000), with little effort to determine malaria infection rates in mosquitoes. Anopeheles sinensis is the predominant anopheline mosquito encountered in the northern part of the ROK (Strickman et al. 2000) and is a known malaria vector (Ree et al. 1967). Other potential vectors include An. lesteri Baisas & Hu and An. yatsushiroensis Miyazaki (Strickman et al. 2000). The taxonomy of An. sinensis and An. lesteri in the ROK is poorly studied. The purpose of the current study was to assess natural Plasmodium vivax infection rates in anopheline mosquitoes collected at U.S. military installations located near the DMZ (high-risk) and other sites throughout the Peninsula. Materials and Methods Study Sites. Anopheline mosquitoes were collected from 29 military bases located throughout the ROK (Table 1). Mosquitoes were evaluated from collections made from August to September 1999 and June to September 2000. Sampling. New Jersey light traps (model 1112, John W. Hock, Gainesville FL) were used for ⬎95% of the trap nights. Insects were captured in a 1-pint polypropylene jar containing a 6 by 6-cm piece of dichlorvos impregnated vinyl strip used as a killing agent. The
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COLEMAN ET AL.: CIRCUMSPOROZOITE PROTEIN IN KOREAN ANOPHELINES
Table 1. Summary of anopheline mosquitoes collected in CY 2000 from a variety of bases throughout the Republic of Korea Location
Females collected
Trap Mean no./ nights trap/night
Tested by ELISA (% of total)
Northern sector (Near DMZ) Munson area Camp Howze Warrior Base Camp Bonifas Camp Giant Camp Greavesa Dongducheon area Camp Casey Camp Hovey Camp Castle Camp Mobile Camp Nimble Korean Training Center Uijongbu area Camp Red Cloud Camp Essayons Camp Kyle Camp Stanley Camp Page
1,395 5,981 2,294 234 9,187
42 46 44 39 N/A
33.2 130.0 52.1 6.0 N/A
1,385 (99.2) 5,675 (94.9) 2,249 (98.0) 227 (97.0) 9,187 (100.0)
172 64 204 153 106 364
86 43 43 43 43 86
2.0 1.5 4.7 3.6 2.5 4.2
172 (100.0) 61 (95.3) 199 (97.5) 152 (99.3) 105 (99.1) 364 (100.0)
34 27 11 232 37
33 33 33 33 108
1.0 0.3 0.3 7.0 0.3
33 (97.1) 27 (100.0) 11 (100.0) 219 (94.4) 34 (91.7)
Yongsan Garrison Camp Humphreys Camp Long Camp Eagle Suwon Air Base Osan Air Base
27 3,062 52 61 152 17
Camp Carroll Camp Hialeah Camp Walker Camp Henry Kunsan Air Base Kwangju Air Base Taegu Air Base Total
74 1 0 0 243 2,786 5 26,900
Central sector 78 0.3 308 9.9 19 2.7 19 3.2 111 1.4 64 0.3
17 (63.0) 3,043 (99.4) 47 (90.4) 53 (86.9) 129 (84.7) 14 (82.4)
Southern sector 66 1.1 68 (95.8) 93 ⬍0.1 1 (100.0) 8 0.0 0 (NA) 8 0.0 0 (NA) 78 3.1 239 (98.4) 170 16.4 2,786 (100.0) 17 0.3 5 (100.0) 1,794 15.0 26,502 (98.5)
a Mosquitoes at Camp Greaves were collected during the course of an evaluation of the efÞcacy of traps for anopheline mosquito surveillance.
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Table 2. Summary of anopheline mosquitoes from the Republic of Korea (CY 1999 and 2000) tested by ELISA for Plasmodium vivax
Site
Total no. of mosquitoes collected at each site (% of site total) An. sinensis/ An. A. lesteri yatsushiroensis sineroides
Camp Bonifas 2,396 (98.6) Camp Greaves 7,156 (71.8) Camp Howze 1,549 (98.9) Camp Humphreys 3,040 (99.9) Warrior Base 6,031 (99.6) Kwangju AFB 2,784 (99.9) All other sites 2,409 (98.7) Totals 25,365 (89.7)
31 (1.3) 2,790 (28.0) 17 (1.1) 3 (0.1) 23 (0.4) 1 (⬍0.1) 25 (1.0) 2,890 (10.2)
Total
3 (0.1) 2,430 19 (0.2) 9,965 0 (0.0) 1,566 0 (0.0) 3,043 0 (0.0) 6,054 1 (⬍0.1) 2,787 8 (0.3) 2,442 31 (0.1) 28,286
Detachment for identiÞcation. Mosquitoes collected in other traps were placed in shipping containers over dry ice and transported after each trap night to the 5th Medical Detachment Entomology Laboratory where they were separated, counted, and identiÞed using the keys from Lee (1998). Anopheles mosquitoes were sorted by species and date of collection and sent to the Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand, to determine malaria infection rates. Anopheles sinensis and An. lesteri were separated using characters from current keys; however, a high percentage of these mosquitoes could not be differentiated using published keys. Therefore, these two species are reported here as An. sinensis/ lesteri. To avoid fungal contamination, all mosquitoes were stored dried before testing. Plasmodium vivax circumsporozoite (CS) protein detection. Mosquitoes from a single trap on a given day were sorted by species and placed in pools of up to 10 individuals. Mosquitoes were tested for the presence of P. vivax 210 and P. vivax 247 CS antigen using the procedures of Wirtz et al. (1985, 1992). Results
New Jersey light trap used a 25-W incandescent bulb and was operated so the light source was 2 m above the ground. Additional traps used to collect mosquitoes included the following: (1) a Shannon trap using a propane lantern (model 5152 D700T, Coleman, Wichita, KS) and human bait as attractants; (2) an American Biophysics Corporation (ABC) light trap (TrapkitDI, ABC, East Greenwich, RI) baited with dry ice; (3) an ABC light trap (Trapkit1) baited with CO2 gas (500 ml/min) dispensed from a 9-kg compressed gas cylinder (Flowkit1, ABC); (4) an ABC Counter ßow geometry (CFG) trap with CO2 alone or with CO2 and octenol; and (5) an ABC Mosquito Magnet trap (Promodel, ABC) with octenol. Details on each of these traps can be found in Burkett et al. (2001). These additional traps (used during a 2-wk period in June and again in September) accounted for nearly onethird of the mosquitoes tested. Trap contents from the New Jersey light traps were submitted weekly in sealed plastic bottles (250 ml) to the 5th Medical
A total of 26,900 anopheline mosquitoes was collected from 29 U.S. military installations during 2000 (Table 1). Of these, 26,502 (98.5%) were tested by enzyme-linked immunosorbent assay (ELISA) (Table 1). An additional 1,784 anopheline mosquitoes collected during AugustÐSeptember 1999 were also tested for malaria. Altogether, 28,286 anopheline mosquitoes (1,784 collected in CY 1999 and 26,502 in CY 2000) were tested by ELISA (Table 2). Of the mosquitoes tested, 89.7% (25,365) were An. sinensis/lesteri, 10.2% (2,890) were An. yatsushiroensis, and 0.1% (31) were An. sineroides Yamada. 66.8% (18,882) of the mosquitoes were collected in New Jersey light traps, whereas 33.2% (9,465) were collected in a variety of other types of traps. The majority of the anophelines in these “other traps” were collected during the course of a mosquito trap evaluation (Burkett et al. 2001) during late June and early September, 2000. Details on the mosquitoes collected in these other traps can be found in Burkett et al. (2001).
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JOURNAL OF MEDICAL ENTOMOLOGY
Of 3,066 pools of mosquitoes tested by ELISA, only two pools (one with nine mosquitoes and one with 10 mosquitoes) of Anopheles sinensis/lesteri mosquitoes were positive for P. vivax CS protein. Both pools were positive for P. vivax 247 only, and not for P. vivax 210. Mosquitoes from both positive pools were collected in a New Jersey light trap: one on 9 September 1999 at Camp Howze, and one on 13 September 1999 at Camp Bonifas. Minimum infection rates in An. sinensis/lesteri mosquitoes collected on each of these dates were 2.9% (1/35) and 3.4% (1/29), respectively. Discussion Personnel from the Department of Public Works (Eighth United States Army), several Preventive Medicine Detachments, the 168th Medical Battalion (Area Support), and the Preventive Medicine Services of the Second Infantry Division conduct routine mosquito surveillance at 29 U.S. installations throughout the Korean Peninsula (Kim et al. 1997, 1999). Data from these collections are used to identify trends in population densities, which in turn are used as a basis for instituting control measures. Before 1993 there was no requirement to evaluate mosquitoes for malaria, because malaria had been eradicated from the ROK in the late 1970s (Paik et al. 1988). Since 1993, when malaria was reintroduced into the ROK, malaria has become Þrmly established along the southern boundary of the DMZ and has subsequently spread to other parts of the peninsula. Due to the long latency period (5- to 13-mo delay in onset of symptoms after the initial infection) in ⬇75% of infections with a North Korean strain of P. vivax (Tiburskaja and Vrublevskaja 1977), it is difÞcult to determine the location where transmission actually occurred. Mosquito collections are therefore concentrated at sites within and near suspected transmission foci. Data on mosquito infection rates are used to institute vector control and place increased emphasize on personal protective measures (e.g., wearing the Battle Dress Uniform with the sleeves rolled down, impregnating the uniform with permethrin, and using deet repellent during periods of risk). In addition, high infection rates in mosquitoes may be used to justify the increased use of chemoprophylaxis as a means of conserving military readiness in areas at high-risk for malaria. During 1999, malaria transmission was suspected near the Joint Security Area (JSA), with a number of cases reported in soldiers deployed to this area. The identiÞcation of one infected mosquito at Camp Greaves (proximal to the JSA) was therefore not a surprise. However, the collection of a Plasmodiuminfected mosquito at Camp Howze was alarming, because transmission of malaria that far south was not suspected. Although the total number of infected mosquitoes was low (⬍0.1% of pools were positive), the minimum infection rates at Camp Greaves (3.4%) and Camp Howze (2.9%) were higher than anticipated. During 2000, the malaria rate in U.S. personnel (U.S. soldiers and Korean Augmentees to the U.S. Army) deployed to the ROK decreased (16 cases diagnosed
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in the ROK compared with ⬎20 cases in each of the previous 2 yr). This decrease may have resulted from an increase in the number of military personnel using malaria chemoprophylaxis. Additionally, the interval between onset of symptoms, clinical diagnosis and initiation of chemotherapy was reduced, thereby reducing the period that infected individuals were able to infect mosquitoes. The number of mosquitoes collected at most installations was also relatively low (Table 1), but this may be a result of trap inefÞciency. The combination of these factors may have contributed to our failure to detect P. vivax in over 26,000 anopheline mosquitoes collected during 2000. Also, mosquitoes collected south of Seoul would not be expected to be infected with malaria because transmission rates in these areas are presumably very low. Current emphasis in the ROK is on expansion of mosquito collections by U.S. forces, particularly in training sites where soldiers are at greater exposure to bites and risk of transmission than while in garrison. Newer trap technologies that maximize the collection of older, epidemiologically important female mosquitoes will be employed at these training sites so that sufÞcient anophelines will be collected to provide more reliable data required for the characterization of malaria transmission in the ROK. Acknowledgments We are extremely thankful for the assistance of Song Chu Yi and her personnel for providing logistical support for the New Jersey light trap collections. We are indebted to Ronald Hamilton (168th Medical Battalion, 18th Medical Command) for his support. We also thank William Herman (5th Medical Detachment), Kenneth McPherson (38th Medical Detachment), and McKinley Rainey (Preventive Medicine Services, 2nd Infantry Division) for providing personnel and logistical support for the collection and identiÞcation of mosquitoes. We also thank Hae Wol Cho (Department of Viral Diseases, Korean National Institute of Health) and colleagues for their support during the special mosquito collections made during mosquito trap evaluations.
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