Density-Dependent Oviposition by Female Aedes albopictus (Diptera: Culicidae) Spreads Eggs Among Containers During the Summer but Accumulates Them in the Fall.

When possible, oviparous females should deposit eggs in sites that maximize the future performance of their offspring. Therefore, studies of oviposition behavior may uncover parameters important to offspring fitness. Gravid female Aedes albopictus (Skuse) were given a choice of containers with leaf infusion or plain water, either open or with a cover with a small opening, and their behavior was compared under summer (long day, higher temperature) or fall (short day, lower temperature) regimes. Open containers with leaf infusion were always preferred, but over time, summer females expanded their choices to oviposit in all containers and follow-up experiments indicated that the number of eggs laid was inversely correlated to the number of eggs present. In contrast, fall females laying diapausing eggs that do not hatch until the spring, accumulated eggs in open containers with food resulting in high egg densities. Combined, these results demonstrate a seasonal shift that suggests either high winter egg mortality or safety in numbers. It also demonstrates that female Ae. albopictus change their behavior based on cues associated with expected added risk, which varies across time and space. The wide distribution of summer eggs across container types may contribute to the fast expansion of Ae. albopictus across its invasive range, but egg accumulation in the fall may be exploited for control.

Insect repellents and associated personal protection for a reduction in human disease.

Personal protection measures against biting arthropods include topical insect repellents, area repellents, insecticide-treated bednets and treated clothing. The literature on the effectiveness of personal protection products against arthropods is mainly limited to studies of prevention of bites, rather than prevention of disease. Tungiasis was successfully controlled by application of topical repellents and scrub typhus was reduced through the use of treated clothing. Successful reduction of leishmaniasis was achieved through the use of topical repellents, treated bednets and treated clothing in individual studies. Malaria has been reduced by the use of insecticide-treated bednets (ITN), certain campaigns involving topical repellents, and the combination of treated bednets and topical repellents. Although area repellents such as mosquito coils are used extensively, their ability to protect humans from vector-transmitted pathogens has not been proven. Taken together, the literature indicates that personal protection measures must be used correctly to be effective. A study that showed successful control of malaria by combining treated bednets and topical repellents suggests that combinations of personal protection measures are likely to be more effective than single methods. Implementation of successful programmes based on personal protection will require a level of cooperation commonly associated with other basic societal functions, such as education and food safety.

Wicking assay for the rapid detection of Rift Valley fever viral antigens in mosquitoes (Diptera: Culicidae).

Rift Valley fever virus (RVFV) causes outbreaks of severe disease in domestic ungulates as well as humans in Africa. There is a logical concern that RVFV could be introduced into the Americas and cause significant health and economic damage based on the precedent of the introduction and spread of West Nile virus (WNV). Unfortunately, there are currently no licensed diagnostic assays available for RVFV in the Americas. In this work, we report on the ability of a novel dipstick assay, VectorTest RVFV antigen assay, modeled on the VecTest assay for WNV, to detect a RVFV-infected female within a pool of mosquitoes. The dipsticks provided results in <20 min, were easy to use, and did not require a laboratory with containment facilities. Although readily able to detect a mosquito with a disseminated RVFV infection, it only occasionally detected RVFV in a mosquito with a nondisseminated infection, and therefore may fail to detect some pools that actually contain one or more positive mosquitoes. The RVFV dipstick assay was highly specific and did not react with samples to which had been added yellow fever, West Nile, Venezuelan equine encephalitis, sandfly fever Naples, sandfly fever Sicilian, or sandfly fever Toscana viruses. The RVFV assay can provide a rapid, safe, easy-to-use assay to alert public health personnel to the presence of RVFV in mosquitoes. Results from this assay will allow a rapid threat assessment and the focusing of vector control measures in high-risk areas.

A live attenuated recombinant dengue-4 virus vaccine candidate with restricted capacity for dissemination in mosquitoes and lack of transmission from vaccinees to mosquitoes.

2Adelta30 is a live dengue-4 virus vaccine candidate with a 30-nucleotide deletion in its 3'-untranslated region. To assess the transmissibility of 2Adelta30 by mosquitoes, we compared its in vivo replication in mosquitoes with that of its wild type DEN-4 parent. Both the vaccine candidate and wild type virus were equally able to infect the mosquito Toxorhynchites splendens after intrathoracic inoculation. Relative to its wild type parent, 2Adelta30 was slightly restricted in its ability to infect the midgut of Aedes aegypti mosquitoes fed on an artificial blood meal and was even more restricted in its ability to disseminate from the midgut to the salivary glands. Thus, the 30-nucleotide deletion rendered the vaccine candidate more sensitive than its wild type parent to the mosquito midgut escape barrier. Most significantly, 2Adelta30 was not transmitted to 352 Ae. albopictus mosquitoes fed on 10 vaccinees, all of whom were infected with the vaccine candidate.

Soldier acceptability of a camouflage face paint combined with DEET insect repellent.

Under operational conditions, current doctrine requires separate application of the standard military insect repellent and camouflage face paint. Working with an industrial partner, Amon Re, Inc., we developed a combined camouflage face paint and insect repellent that offers excellent protection from arthropod disease vectors. We undertook a study to determine whether the new product was acceptable to soldiers under field conditions. A new formulation of camouflage face paint containing the insect repellent N,N-diethyl-3-methyl-benzamide (DEET) was tested for user acceptability during a joint-service, multinational, military field training exercise (Operation Cobra Gold 1999) in Thailand. Soldiers testing the camouflage face paint were members of one of three companies (A, B, and C) of 1st Battalion, 21st Infantry Regiment, 25th Infantry Division. Soldiers in A company (N = 98) received standard military camouflage face paint and the U.S. military's Extended Duration Topical Insect and Arthropod Repellent containing 33% DEET, soldiers in B company (N = 75) received a new formulation of camouflage face paint without DEET, and soldiers in C company (N = 88) received the new formulation of camouflage face paint containing 30% DEET. Every soldier who volunteered to participate completed the study and submitted responses to the questionnaire. The different treatments were evaluated while soldiers spent 4 days conducting simulated combat exercises in a hot, tropical environment in central Thailand. Soldiers were provided the test materials, given a briefing on the study, and completed an initial questionnaire on May 19, 1999. Soldiers completed a final questionnaire after they returned from the training exercise on May 24, 1999. Results of the study indicated that soldiers found the new formulation of 30% DEET camouflage face paint easier to apply (88% of respondents) and remove (77%) than the current standard issue camouflage face paint. Soldiers liked the new 30% DEET camouflage face paint formulation better (61%) and were more likely to recommend it (67%) than the old camouflage face paint formulation. Although the addition of DEET to the camouflage face paint occasionally caused minor irritation after application for a short period (less than 15 minutes), the new camouflage face paint formulation with 30% DEET was evaluated most often (72%) as either good or excellent. The new 30% DEET camouflage face paint formulation offers significant operational advantages: protection from disease-transmitting arthropods, elimination of the need to carry separate insect repellent and camouflage face paint, and reduction in the time required to apply repellent and camouflage face paint separately.

Field evaluation of deet and a piperidine repellent against Aedes communis (Diptera: Culicidae) and Simulium venustum (Diptera: Simuliidae) in the Adirondack Mountains of New York.

Repellent efficacy of N,N-diethyl-3-methyl-benzamide (deet), the piperidine, 1-[3-cyclohexen-1-ylcarbonyl]-2-methylpiperidine (AI3-37220), and a 1:1 ratio of deet + AI3-37220 were evaluated topically (0.25 mg/cm2 applied in ethanol solution) on human volunteers against the mosquito Aedes communis (DeGeer) and the black fly Simulium venustum Say. The average repellency of all three formulations was > 95% at 4 h. For both mosquitoes and black flies, deet alone provided < 90% protection at 6 h, whereas AI3-37220 provided > 95% protection. Although repellent treatments were not significantly different overall, the contrasts between AI3-3720 versus deet were significant at 6 and 8 h. The 95% confidence interval on percent repellency at 6 h ranged from 90.1 to 98.9% for AI3-37220 versus 64.3 to 82.2% for deet, and at 8 h ranged 76.1 to 88.5% for AI3-37220 versus 47.8 to 64.0% for deet. Similarly, the confidence interval for protection against black flies at 6 h by (AI3-37220 ranged from 86.3 to 99.5% and did not overlap with the confidence interval provided by deet alone (51.2 to 78.8%). There was no evidence of synergistic repellency from a combination of the two compounds; i.e., protection from combined compounds was no better than either repellent used alone.

Mosquito collections following local transmission of Plasmodium falciparum malaria in Westmoreland County, Virginia.

A 63-year-old woman from Colonial Beach, Westmoreland County, VA, was diagnosed with Plasmodium falciparum malaria on July 19, 1998. The woman had no history of international travel, intravenous drug use, blood transfusion, or other risk factor for contracting the disease. She seldom left the county and generally spent her evenings indoors, leading to the conclusion that she had been bitten locally by an infected mosquito. Colonial Beach is host to a population of migrant agricultural laborers from areas in which malaria occurs, but a blood survey of 89 Haitians and Mexicans failed to find Plasmodium parasites, specific antibodies, or clinical cases of malaria. Mosquito surveys were conducted during 2 days (July 22 and 28, 1998) with carbon-dioxide-baited light traps, larval and pupal collections, and landing collections. Thirteen species of mosquitoes were identified morphologically, including 4 potential vectors: Anopheles crucians, An. punctipennis, An. smaragdinus (new state record), and An. quadrimaculatus s.s. (new state record). Identifications of the latter 2 species were confirmed by sequencing of the ITS2 DNA region from adults reared from locally collected larvae. Anopheles smaragdinus was the most common biting species among the potential vectors, although An. crucians was the most abundant in other kinds of collections. In addition, Ae. albopictus was collected in Westmoreland County for the 1st time.

Doxycycline and rifampicin for mild scrub-typhus infections in northern Thailand: a randomised trial.

BACKGROUND: Some strains of scrub typhus in northern Thailand are poorly responsive to standard antirickettsial drugs. We therefore did a masked, randomised trial to compare rifampicin with standard doxycycline therapy for patients with scrub typhus. METHODS: Adult patients with strictly defined, mild scrub typhus were initially randomly assigned 1 week of daily oral treatment with 200 mg doxycycline (n=40), 600 mg rifampicin (n=38), or doxycycline with rifampicin (n=11). During the first year of treatment, the combined regimen was withdrawn because of lack of efficacy and the regimen was replaced with 900 mg rifampicin (n=37). Treatment outcome was assessed by fever clearance time (the time for oral temperature to fall below 37.3 degrees C). FINDINGS: About 12,800 fever patients were screened during the 3-year study to recruit 126 patients with confirmed scrub typhus and no other infection, of whom 86 completed therapy. Eight individuals received the combined regimen that was discontinued after 1 year. The median duration of pyrexia was significantly shorter (p=0.01) in the 24 patients treated with 900 mg daily rifampicin (fever clearance time 22.5 h) and in the 26 patients who received 600 mg rifampicin (fever clearance time 27.5 h) than in the 28 patients given doxycycline monotherapy (fever clearance time 52 h). Fever resolved in a significantly higher proportion of patients within 48 h of starting rifampicin (900 mg=79% [19 of 24], 600 mg=77% [20 of 26]) than in patients treated with doxycycline (46% [13 of 28]; p=0.02). Severe gastrointestinal events warranted exclusion of two patients on doxycyline. There were two relapses after doxycycline therapy, but none after rifampicin therapy. INTERPRETATION: Rifampicin is more effective than doxycycline against scrub-typhus infections acquired in northern Thailand, where strains with reduced susceptibility to antibiotics can occur.

Mosquito surveillance in the Demilitarized Zone, Republic of Korea, during an outbreak of Plasmodium vivax malaria in 1996 and 1997.

Since 1993, more than 2,000 cases of vivax malaria have occurred in the Republic of Korea in an epidemic that ended nearly 20 malaria-free years. Most malaria has occurred in the northwestern part of the country, mainly affecting Korean military personnel. As a part of an operational surveillance effort, we sampled mosquitoes in and near the Demilitarized Zone (Paju County, Kyonggi Province) during the last 2 wk of July in 1996 and from May 15 to September 10 in 1997. The 1st year, landing collections were done at 5 different sites; the 2nd year, carbon-dioxide-baited light traps at 5 sites, larval collections in 10 adjacent fields, and landing collections at 1 site in the Demilitarized Zone were performed weekly. Of 17 species collected, Anopheles sinensis was consistently the most abundant mosquito, comprising 79-96% of mosquitoes. The diel pattern of biting by An. sinensis varied by location and season, with the majority of individuals biting late at night during warm weather (>20 degrees C) and early at night during cool weather. In contrast, Aedes vexans nipponii (the 2nd most abundant species) bit in the greatest numbers at the same time all season, from 2000 to 2300 h. Among the correlates with abundance of An. sinensis were average nighttime temperature 2 wk previous to the night in question, wind late at night (negatively correlated), and apparent size of the moon (negatively correlated). The data showed that the exact number of An. sinensis biting could not be estimated from numbers collected in carbon-dioxide-baited light traps. On the other hand, a threshold of 15 An. sinensis per trap night corresponded (88% accuracy) to a threshold of 12 mosquitoes biting 2 adjacent collectors per night. Larval collections were also significantly correlated with landing collections, despite inexact sampling methods and separation of the larval habitat from the site where landing collections were performed. Operational entomology assets using nighttime temperature records, carbon-dioxide-baited light traps, and larval collections should be able to target their efforts in Korea more efficiently.

Effects of magnetic fields on mosquitoes.

Phylogenetically diverse organisms, including some insects, are able to detect and respond to magnetic fields comparable to the Earth's magnetic field. Because of their tremendous importance to public health, mosquitoes were tested for the presence of remanent ferromagnetic material indicative of a biological compass and also tested for behavioral responses to magnetic fields. Using a superconducting quantum interferometry device, we found that significant remnant was probably due to attraction of ferromagnetic dust onto the surface of live or dead mosquitoes. Most mosquitoes placed in a 1.0-gauss, uniform magnetic field moved until they were oriented parallel to the field. Two of 3 species of mosquitoes tested took fewer blood meals in a rotating magnetic field than in the Earth's normal magnetic field.

Longitudinal studies of Aedes aegypti (Diptera: Culicidae) in Thailand and Puerto Rico: blood feeding frequency.

We used a histologic technique to study multiple blood feeding in a single gonotrophic cycle by engorged Aedes aegypti (L.) that were collected weekly for 2 yr from houses in a rural village in Thailand (n = 1,891) and a residential section of San Juan, Puerto Rico (n = 1,675). Overall, mosquitoes from Thailand contained significantly more multiple meals (n = 1,300, 42% double meals, 5% triple meals) than mosquitoes collected in Puerto Rico (n = 1,156, 32% double meals, 2% triple meals). The portion of specimens for which frequency of feeding could not be determined was 31% at both sites. We estimated that on average Ae. aegypti take 0.76 and 0.63 human blood meals per day in Thailand and Puerto Rico, respectively. However, frequency of multiple feeding varied among houses and, in Puerto Rico, the neighborhoods from which mosquitoes were collected. In Thailand 65% of the mosquitoes fed twice on the same day, whereas in Puerto Rico 57% took multiple meals separated by > or = 1 d. At both sites, the majority of engorged specimens were collected inside houses (Thailand 86%, Puerto Rico 95%). The number of blood meals detected was independent of where mosquitoes were collected (inside versus outside of the house) at both sites and the time of day collections were made in Puerto Rico. Feeding rates were slightly higher for mosquitoes collected in the afternoon in Thailand. Temperatures were significantly higher and mosquitoes significantly smaller in Thailand than in Puerto Rico. At both sites female size was negatively associated with temperature. Rates of multiple feeding were associated positively with temperature and negatively with mosquito size in Thailand, but not in Puerto Rico. Multiple feeding during a single gonotrophic cycle is a regular part of Ae. aegypti biology, can vary geographically and under different climate conditions, and may be associated with variation in patterns of dengue virus transmission.

Longitudinal studies of Aedes aegypti (Diptera: Culicidae) in Thailand and Puerto Rico: population dynamics.

Aspiration collections of adult Aedes aegypti (L.) were made weekly from inside and outside of houses for 3 yr in a rural Thai village (n = 9,637 females and n = 11,988 males) and for 2 yr in a residential section of San Juan, Puerto Rico (n = 5,941 females and n = 6,739 males). In Thailand, temperature and rainfall fell into distinct seasonal categories, but only temperature was correlated with fluctuations in female abundance. Average weekly temperature 6 wk before mosquitoes were collected and minimum weekly temperature during the week of collection provided the highest correlations with female abundance. Accounting for annual variation significantly improved Thai models of temperature and mosquito abundance. In Puerto Rico, temperature, but not rainfall, could be categorized into seasonal patterns. Neither was correlated with changes in female abundance. At both sites the vast majority of females were collected inside houses and most contained a blood meal. Most teneral females were collected outside. Wing length--an indicator of female size--and parity, egg development or engorgement status were not correlated, indicating that feeding success and survival were not influenced by female size. At both sites, females fed almost exclusively on human hosts (> or = 96%), a pattern that did not change seasonally. In Puerto Rico more nonhuman blood meals were detected in mosquitoes collected outside than inside houses; no such difference was detected in Thailand. Gut contents of dissected females indicated that females in the Thai population had a younger age distribution and fed more frequently on blood than did Ae. aegypti in Puerto Rico. Our results indicated that aspects of this species' biology can vary significantly from one location to another and 1 yr to the next.

Distribution of dengue and Japanese encephalitis among children in rural and suburban Thai villages.

In the rainy season of 1989, IgG and IgM antibodies against dengue and Japanese encephalitis viruses (measured by enzyme-linked immunoassay [ELISA]) in serum from all primary-school children in two areas of central Thailand were sampled in order to choose a study site for more detailed epidemiological and entomological analysis. Students in three schools in the largely non-agricultural, suburban community of Bang Bua Thong, Nontaburi Province were sampled in late June and July. Of 1,477 children, 33/1,000 had recent dengue infection and 7/1,000 had recent JE infection. The rate of dengue infection in each village influenced the rate in schools, in that the rate of the school could be predicted from the proportion of students coming from each village. This result suggested that most transmission occurred in the residential environment; otherwise, the rate in each village going to a single school would be identical. Serum samples were taken in late August in the agricultural community of Hua Samrong, Chachoengsao Province. Of 748 students in two schools, 95/1,000 had signs of recent dengue infection and 32/1,000 had signs of recent JE infection. Two of 12 villages had significantly less flavivirus infection than some other villages and three villages had significantly more flavivirus infection. The children from one village had a dengue infection rate of 256 per 1,000, which was higher than the national average for the worst year (1987) previously recorded in Thailand. Within Hua Samrong, there was evidence for significant dengue transmission in one of the schools and concentrated transmission in small areas of two of the villages. The younger age group (3-8 years old) had significantly higher risk of infection by either flavivirus than older children. Elevated homes with wooden floors had significantly higher risk of dengue in the largest village. The observations from 1989 describe the epidemiological situation in rapidly developing, rural villages. This stage of development is probably being repeated throughout Southeast Asia as formerly isolated, rural villages become connected by transportation and economy to urban centers. What appears to be a single dengue outbreak based on passive surveillance conducted on a regional basis may actually be a variety of epidemiological situations. The practical implication of this conclusion is that application of a combination of vaccination and vector control should be targeted to higher risk areas in order to increase the likelihood of regional dengue virus eradication.

Serologic responses of Korean soldiers serving in malaria-endemic areas during a recent outbreak of Plasmodium vivax.

Anti-Pv200 antibody levels were assessed in samples from endemic areas of Plasmodium vivax malaria in the Republic of Korea (ROK), using an indirect enzyme-linked immunosorbent assay (ELISA) method. Asymptomatic carriers of P. vivax were detected using nested polymerase chain reaction (PCR) of blood samples. Anti-Pv200 antibody levels in 20 vivax malaria patients (optical density +/- standard deviation [OD +/- SD] values 1.85 +/- 0.29 of IgG isotype and 1.33 +/- 1.33 of IgM isotype) were markedly higher than those of uninfected, malaria-naive controls (0.08 +/- 0.16 of IgG isotype and 0.04 +/- 0.04 of IgM isotype). Antibody levels for 7 out of 8 soldiers with a recent malaria infection were sustained above the cut-off values for 4 months after successful treatment. Analysis of serum collected from 40 healthy, asymptomatic soldiers who had a P. vivax malaria attack within 3 months after our sampling, revealed 11 antibody-positive samples (27.5%), compared to 5 positive samples (12.5%) collected from a random selection of 40 soldiers. Among a larger pool of 1,713 soldiers who had served in high-risk areas for P. vivax transmission, 15% were antibody positive. Among 1,000 blood samples from asymptomatic soldiers who had served in the high-risk areas, 4 samples (0.4%) were parasite positive, as determined by nested PCR. Our results show that anti-Pv200 antibody levels can provide useful information in the late diagnosis of P. vivax malaria infection in a previously naive population and also in large seroepidemiologic studies. Furthermore, our results suggest that asymptomatic P. vivax carriers could be important in the current outbreak of malaria in Korea.

Frequency of Wolbachia infection in laboratory and field sand fly (Diptera: Psychodidae) populations.

Using a polymerase chain reaction (PCR) assay with primers designed for the Wolbachia 16S rRNA and outer surface protein (wsp) gene, we screened 11 laboratory colonies and 4 field samples of 6 sand fly species for Wolbachia infection. Infection was only detected in 3 laboratory colonies of Phlebotomus papatasi Scopoli, originally collected in Israel, Egypt, and Saudi Arabia.

Evaluation of the malaria threat at the multipurpose range complex, Yongp'yong, Republic of Korea.

As part of an operational effort to control the transmission of malaria after its reemergence in the Republic of Korea, a number of U.S. military and Korean civilian health workers assessed the situation at a large training site where two cases had been transmitted. The vector mosquitoes (Anopheles sinensis) probably had a limited flight range (< 1 km) in this situation based on low numbers in permanent light traps and biting collections at the billeting area compared with collections from light traps and larval surveys where the mosquito was abundant. The threat of malaria was proven by the presence of infected local Korean residents (13 of 105 sampled). However, only a small proportion of U.S. soldiers (39 of 78 surveyed) at the training site used personal protective measures. Further efforts should include improved techniques for informing and motivating soldiers to use personal protective measures and cooperation with Korean authorities to perform vector control in civilian areas bordering the training area.

Laboratory evaluation of AI3-37220, AI3-35765, CIC-4, and deet repellents against three species of mosquitoes.

Four repellents, N,N-diethyl-3-methyl-benzamide (deet), 2-hydroxy-methyl-cyclohexyl acetic acid lactone (CIC-4), and 2 piperidines (1-[3-cyclohexen-1-ylcarbonyl] piperidine [AI3-35765] and 1-[3-cyclohexen-1-ylcarbonyl]-2-methylpiperidine [AI3-37220]) were evaluated alone and in combination against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus using a modified in vitro test system. This method was a valuable tool for comparing effective concentrations of the new compounds. Because of the controlled conditions of the test, it was possible to use the results of assays that had been conducted over a 5-year period and to perform the many replications necessary to evaluate combinations of compounds. The new candidate repellents were generally as effective as deet. Although speculative at this time, there was some evidence of synergistic interaction. Repellent combinations of CIC-4/AI3-37220/AI3-35767, deet/AI3-35765, and deet/AI3-37220/AI3-35765 against An. stephensi and CIC-4/AI3-35765, deet/AI3-37220/AI3-35765, AI3-37220/AI3-35765, and CIC-4/AI3-37220 against Ae. aegypti were more effective than the component compounds alone.

Response to chloroquine of Plasmodium vivax among South Korean soldiers.

The response to standard chloroquine treatment was evaluated, by microscopical examination of blood-smears, among 81 soldiers diagnosed with Plasmodium vivax malaria in South Korea in 1996. The smears were prepared pre-treatment and 3, 14 and 28 days after starting chemotherapy. Parasitaemias were determined after staining the smears with Giemsa's stain. Blood samples from the patients who were not smear-negative by day 3 were carefully checked for parasites, by staining smears with Acridine Orange and by a PCR-based assay. Only two of the patients appeared to be parasitaemic on day 14 and were therefore considered treatment failures. Although both were apparently cured after additional therapy with the same regimen, one had a recurrence 8 months later. Most cases of recent, resurgent malaria in South Korea therefore appear to sensitive to chloroquine.

Recovery and viability of Orientia tsutsugamushi from packed red cells and the danger of acquiring scrub typhus from blood transfusion.

BACKGROUND: The purpose of this study was to determine whether infective Orientia tsutsugamushi, the etiologic agent of scrub typhus, could survive normal blood banking processing and storage procedures. STUDY DESIGN AND METHODS: Mononuclear cells isolated from whole blood by density gradient centrifugation were inoculated with O. tsutsugamushi, Karp strain. Infection of the mononuclear cells was confirmed by Giemsa stain, direct fluorescent antibody assay, and polymerase chain reaction using primers specific for the groESL operon of O. tsutsugamushi. The quantity of rickettsial particles in each preparation was determined by direct counts from the Giemsa-stained preparations. Infected mononuclear cells were returned to their respective aliquots of packed red blood cells, which were then either stored at 4 degrees C or glycerolized and frozen at -70 degrees C. RESULTS: Rickettsiae survived up to 10 days (but not 30 days) of refrigerated storage and 45 days of frozen storage, as determined by inoculation of mice with 0.5-mL aliquots of the blood components. Infection of the mice was determined by illness, death, direct fluorescent antibody assay of peritoneal smears, polymerase chain reaction of blood, and enzyme-linked immunosorbent assay detection of antibodies in plasma. CONCLUSION: Because the quantity of rickettsiae injected into the mice was comparable to the quantity reported in the literature for human blood during natural infections, scrub typhus could present a risk in blood collected from donors in endemic areas. This may especially be true, because people can be rickettsemic before illness, after successful antibiotic treatment, and chronically after resolution of disease.