Direct detection of Leishmania from clinical samples.

The ability to rapidly and accurately diagnose leishmaniasis is a military priority. Testing was conducted to evaluate diagnostic sensitivity and specificity of field-expedient Leishmania genus and visceral Leishmania specific dual-fluorogenic, hydrolysis probe (TaqMan), polymerase chain reaction assays previously established for use in vector surveillance. Blood samples of patients with confirmed visceral leishmaniasis and controls without the disease from Baringo District, Kenya, were tested. Leishmania genus assay sensitivity was 100% (14/14) and specificity was 84% (16/19). Visceral Leishmania assay sensitivity was 93% (13/14) and specificity 80% (4/5). Cutaneous leishmaniasis (CL) skin scrapes of patients from Honduras were also evaluated. Leishmania genus assay sensitivity was 100% (10/10). Visceral Leishmania assay specificity was 100% (10/10) from cutaneous leishmaniasis samples; no fluorescence above background was reported. These results show promise in a rapid, sensitive, and specific method for Leishmania direct detection from clinical samples.

LONGITUDINAL EVALUATION OF MALARIA EPIDEMIOLOGY IN AN ISOLATED VILLAGE IN WESTERN THAILAND: I. STUDY SITE AND ADULT ANOPHELINE BIONOMICS.

This is the first in a series of papers describing the epidemiology of malaria in an isolated village in western Thailand. The study site was the village of Kong Mong Tha, located in Sangkhla Buri District, Kanchanaburi Province, Thailand. In this paper we present an overview of the study site and results from our adult anopheline mosquito surveillance conducted over 56 consecutive months from June 1999 until January 2004. The collection site, indoor/outdoor location, parity, biting activity and Plasmodiumfalciparum (Pf) and P. vivax (Pv) infection rates were used to calculate seasonal entomological inoculation rates for the predominant four Anopheles species. A total of 21,566 anophelines representing 28 distinct species and 2 groups that were not identified to species were collected using human bait, with almost 95% of the collection consisting of Anopheles minimus, An. maculatus, An. sawadwongporni and An. barbirostris/campestris. Mosquitoes generally peaked during the wet season, were collected throughout the night, and were collected most often outside (ca. 75%) versus inside (ca. 25%) of houses. Approximately 50% of collected mosquitoes were parous. Overall Plasmodium infection rates were 0.27%, with a total of 16 and 42 pools of Pf- and Pv-positive mosquitoes, respectively. Annual EIRs were 2.3 times higher for Pv than for Pf, resulting in approximately 5.5 and 2.6 infective bites per person per year, respectively. The results suggest An. minimus and An. maculatus are the primary and secondary vectors of Pf and Pv transmission in Kong Mong Tha, while An. sawadwongporni and An. barbirostris/campestris also appear to play a role based on the presence of circumsporozoite protein (CSP) in the head/thorax of the specimens tested.

Detection of Dengue Virus in Mosquito Extracts and Human Clinical Samples Using a Field Expedient Molecular Platform.

Dengue fever occurs in localized outbreaks and can significantly erode troop strength and mission readiness. Timely identification of dengue virus (DENV) provides for rapid and appropriate patient management decisions, such as medical evacuation and supportive therapies, as well as help to promote Force Health Protection through vector control and personal protective measures. The "Ruggedized" Advanced Pathogen Identification Device is a field-friendly PCR (Polymerase Chain Reaction) platform that can be used to facilitate early identification of DENV. We developed a dry-format PCR assay on this platform. The assay demonstrated 100% analytical specificity for detecting dengue using a cross-reactivity panel. We used a panel of 102 acute, DENV isolation positive serum samples and 25 DENV negative samples; the assay demonstrated a clinical sensitivity of 97.1% (95% C.I. 91.6-99.4%) and specificity of 96.0% (95% C.I. 79.7-99.9%) in identifying patients with dengue infection. We also used the assay to test mosquito homogenates from 28 adult female Aedes aegypti. A single DENV infected mosquito was identified using the PCR assay and confirmed using immunofluorescence as a reference method. Much of the testing was performed under austere field conditions. Together, our results demonstrate the utility of this assay for detecting DENV in vector and human samples in field environments.

Field detection of Tembusu virus in western Thailand by rt-PCR and vector competence determination of select culex mosquitoes for transmission of the virus.

Tembusu virus (TMUV; Ntaya serocomplex) was detected in two pools of mosquitoes captured near Sangkhlaburi, Thailand, as well as from sera from sentinel ducks from the same area. Although TMUV has been isolated from several mosquito species in Asia, no studies have ever shown competent vectors for this virus. Therefore, we allowed mosquitoes captured near Sangkhlaburi to feed on young chickens that had been infected with TMUV. These mosquitoes were tested approximately 2 weeks later to determine infection, dissemination, and transmission rates. Culex vishnui developed high viral titers after feeding on TMUV-infected chicks and readily transmitted virus to naïve chickens. In contrast, Cx. fuscocephala seemed less susceptible to infection, and more importantly, zero of five fuscocephala with a disseminated infection transmitted virus by bite, indicating a salivary gland barrier. These results provide evidence for the involvement of Culex mosquitoes in the transmission of TMUV in the environment.

Leishmania detection in sand flies using a field-deployable real-time analytic system.

We describe here the development and evaluation of advanced vector surveillance analytic technologies for real-time leishmaniasis risk assessment. Leishmania genus and visceral leishmaniasis causative agent--specific dual fluorogenic-probe hydrolysis (TaqMan), thermally stable (freeze-dried) polymerase chain reaction assays were developed using field-durable analytic instrumentation. In laboratory testing with a panel of diverse Leishmania species from culture and infected sand flies, the sensitivity and specificity of both assays were 100% concordant with DNA sequencing. In specificity testing with Leishmania genetic near neighbors, clinically significant organisms, and human genomic DNA, no detectable fluorescence above background was observed. Field evaluation was conducted in southern Iraq using wild sand flies. In field testing, Leishmania genus assay was 100% sensitive and 96% specific with a single false-positive result. The visceral leishmaniasis genotype assay was 100% sensitive and 100% specific compared to DNA sequencing. Thermally stable polymerase chain reaction assays vastly simplified transportation and storage. Assay preparation and analysis required less than 2 hours.

Impact of phlebotomine sand flies on United States military operations at Tallil Air Base, Iraq: 5. Impact of weather on sand fly activity.

In this study, we examined the effect of weather and moon illumination on sand fly activity, as measured by light trap collections made between 2 May 2003 and 25 October 2004 at Tallil Air Base, Iraq. Wind speed, temperature, dew point, percentage of sky cover, and moon illumination were entered into principal components analysis. The resulting principal components were entered into stepwise regression to develop a model of the impact of the weather on sand fly collections. Wind speed, percentage of sky cover, and moon illumination each had a strong inverse relationship with the number of sand flies collected, whereas temperature displayed a direct relationship to sand fly collections. Our data indicate that sand fly light trap catches at Tallil Air Base are highest on warm, clear nights with low wind speed and minimal illumination from the moon.

Impact of phlebotomine sand flies on United State military operations at Tallil Air Base, Iraq: 6. Evaluation of insecticides for the control of sand flies.

We conducted a series of field experiments in 2003 and 2004 to evaluate the efficacy of a variety of insecticides and insecticide application technologies for the control of phlebotomine sand flies at Tallil Airbase, Iraq. During the experiments, 53,263 sand flies were collected. The experiments evaluated the following: (1) routine sand fly control operations using a variety of residual and area-wide insecticides; (2) a combination of five different insecticide application methods in and around tents; (3) residual application of lambda-cyhalothrin and ultra-low volume application of pyrethrins in houses; (4) carbaryl and lambda-cyhalothrin applied as barrier sprays; (5) a deltamethrin-impregnated fence; (6) lambda-cyhalothrin applied as a residual spray in concrete manholes; (7) deltamethrin-treated flooring in tents; and 8) ultra-low volume-applied malathion. Although some of the experiments resulted in limited reductions in the number of sand flies collected in light traps, in no instance did we completely eliminate sand flies or reduce populations for a sustained period. The implications of these findings are discussed.

Anointing chemicals and hematophagous arthropods: responses by ticks and mosquitoes to citrus (Rutaceae) peel exudates and monoterpene components.

Some birds and mammals roll on or wipe themselves with the fruits or leaves of Citrus spp. or other Rutaceae. These anointing behaviors, as with anointing in general, are thought to function in the topical acquisition of chemicals that deter consumers, including hematophagous arthropods. We measured avoidance and other responses by nymphal lone star ticks (Amblyomma americanum) and adult female yellow fever mosquitoes (Aedes aegypti) to lemon peel exudate and to 24 volatile monoterpenes (racemates and isomers), including hydrocarbons, alcohols, aldehydes, acetates, ketones, and oxides, present in citrus fruits and leaves in order to examine their potential as arthropod deterrents. Ticks allowed to crawl up vertically suspended paper strips onto a chemically treated zone avoided the peel exudate and geraniol, citronellol, citral, carveol, geranyl acetate, α-terpineol, citronellyl acetate, and carvone. Ticks confined in chemically treated paper packets subsequently were impaired in climbing and other behaviors following exposure to the peel exudate and, of the compounds tested, most impaired to carveol. Mosquitoes confined in chambers with chemically treated feeding membranes landed and fed less, and flew more, when exposed to the peel exudate than to controls, and when exposed to aldehydes, oxides, or alcohols versus most hydrocarbons or controls. However, attraction by mosquitoes in an olfactometer was not inhibited by either lemon peel exudate or most of the compounds we tested. Our results support the notion that anointing by vertebrates with citrus-derived chemicals deters ticks. We suggest that some topically applied compounds are converted into more potent arthropod deterrents when oxidized on the integument of anointed animals.

Illustrated keys to the mosquitoes of Thailand. VI. Tribe Aedini.

Illustrated keys for the identification of the fourth-instar larvae and adult females of the mosquito species of tribe Aedini in Thailand are presented, along with the geographic distribution of the species and the known habitats of their immature stages. The keys are the first to encompass the recent revisionary studies of tribe Aedini. One hundred and seventy-five species of Aedini belonging to 38 genera and 18 subgenera are recognized in Thailand. Two species of genus Armigeres, two of genus Collessius, and one of genus Downsiomyia are undescribed. Himalaius simlensis [formerly Aedes (Finlaya) simlensis], Hopkinsius (Yamada) albocinctus [formerly Aedes (Finlaya) albocinctus], Downsiomyia nipponica and Downsiomyia saperoi [formerly species of Aedes (Finlaya)], and Hulecoeteomyia pallirostris [formerly Ochlerotatus (Finlaya) pallirostris] are new country records. Aedimorphus (formerly a subgenus of Aedes), Cancraedes, and Rhinoskusea (formerly subgenera of Ochlerotatus) are recognized as genera, and genus Petermattinglyius includes species previously included in Diceromyia (formerly a subgenus of Aedes) in Thailand. Heteraspidion, Huangmyia, Stegomyia, and Xyele are newly recognized subgenera of genus Stegomyia (formerly a subgenus of Aedes), which includes eight species without subgeneric placement. Two unidentified and unplaced species were discovered in Thailand.

Use of vector diagnostics during military deployments: recent experience in Iraq and Afghanistan.

Vector-borne diseases such as malaria, dengue, and leishmaniasis are a threat to military forces deployed outside of the United States. The availability of specific information on the vector-borne disease threat (e.g., presence or absence of a specific disease agent, temporal and geographic distribution of competent vectors, and vector infection rates) allows for effective implementation of appropriate measures to protect our deployed military forces. Vector diagnostics can provide critical, real-time information crucial to establishing effective vector prevention/control programs. In this article we provide an overview of current vector diagnostic capabilities, evaluate the use of vector diagnostics in Operation Enduring Freedom and Operation Iraqi Freedom, and discuss the concept of operations under which vector diagnostics are employed.

Impact of phlebotomine sand flies on U.S. military operations at Tallil Air Base, Iraq: 4. Detection and identification of leishmania parasites in sand flies.

Sand flies collected between April 2003 and November 2004 at Tallil Air Base, Iraq, were evaluated for the presence of Leishmania parasites using a combination of a real-time Leishmania-generic polymerase chain reaction (PCR) assay and sequencing of a 360-bp fragment of the glucose-6-phosphate-isomerase (GPI) gene. A total of 2,505 pools containing 26,574 sand flies were tested using the real-time PCR assay. Leishmania DNA was initially detected in 536 pools; however, after extensive retesting with the real-time PCR assay, a total of 456 pools were considered positive and 80 were considered indeterminate. A total of 532 samples were evaluated for Leishmania GPI by sequencing, to include 439 PCR-positive samples, 80 PCR-indeterminate samples, and 13 PCR-negative samples. Leishmania GPI was detected in 284 samples that were sequenced, to include 281 (64%) of the PCR-positive samples and 3 (4%) of the PCR-indeterminate samples. Of the 284 sequences identified as Leishmania, 261 (91.9%) were L. tarentolae, 18 (6.3%) were L. donovani-complex parasites, 3 (1.1%) were L. tropica, and 2 were similar to both L. major and L. tropica. Minimum field infection rates were 0.09% for L. donovani-complex parasites, 0.02% for L. tropica, and 0.01% for the L. major/tropica-like parasite. Subsequent sequencing of a 600-bp region of the "Hyper" gene of 12 of the L. donovani-complex parasites showed that all 12 parasites were L. infantum. These data suggest that L. infantum was the primary leishmanial threat to U.S. military personnel deployed to Tallil Air Base. The implications of these findings are discussed.

The role of the United States military in the development of vector control products, including insect repellents, insecticides, and bed nets.

Arthropod-borne diseases such as malaria, dengue, scrub typhus, and leishmaniasis continue to pose a significant threat to U.S. military forces deployed in support of operational and humanitarian missions. These diseases are transmitted by a variety of arthropods, including mosquitoes, ticks, chiggers, sand flies, and biting midges. In addition to disease threats, biting arthropods can cause dermatitis, allergic reactions, and sleep loss; therefore, monitoring of vector impact and integrated use of personal protective measures (PPM) and methods to reduce the vector populations are needed to protect service members. The U.S. military has played a vital role in vector identification tools and the development and testing of many of the most effective PPM and vector control products available today, including the topical repellent DEET and the repellent/insecticide permethrin, which is applied to clothing and bed nets. Efforts to develop superior products are ongoing. Although the U.S. military often needs vector control products with rather specific properties (e.g., undetectable, long-lasting in multiple climates) in order to protect its service members, many Department of Defense vector control products have had global impacts on endemic disease control.

Impact of phlebotomine sand flies on U.S. military operations at Tallil Air Base, Iraq: 3. Evaluation of surveillance devices for the collection of adult sand flies.

We evaluated the effectiveness of commercially available light traps and sticky traps baited with chemical light sticks for the collection of phlebotomine sand flies. Evaluations were conducted at Tallil Air Base, Iraq, in 2003. In an initial study, a Centers for Disease Control and Prevention (CDC)-style trap with UV bulb collected significantly more sand flies than did an up-draft CDC trap, a standard down-draft CDC trap (STD-CDC), or a sticky strap with a green chemical light stick. In a subsequent study, we found that the addition of chemical light sticks to sticky traps resulted in a significant increase in the number of sand flies collected compared with sticky traps without the light sticks. These data indicate that 1) the CDC light trap with an UV bulb is an effective alternative to the standard CDC light trap for collecting phlebotomine sand flies in Iraq, and 2) that the addition of a chemical light stick to a sticky trap can result in a field-expedient tool for the collection of sand flies.

Impact of phlebotomine sand flies on U.S. Military operations at Tallil Air Base, Iraq: 2. Temporal and geographic distribution of sand flies.

CDC miniature light traps were used to evaluate the general biology of phlebotomine sand flies from April 2003 to November 2004 at Tallil Air Base, Iraq. Factors evaluated include species diversity and temporal (daily and seasonal) and geographic distribution of the sand flies. In addition, the abundance of sand flies inside and outside tents and buildings was observed. In total, 61,630 sand flies were collected during 1,174 trap nights (mean 52 per trap, range 0-1,161), with 90% of traps containing sand flies. Sand fly numbers were low in April, rose through May, were highest from mid-June to early September, and dropped rapidly in late September and October. More than 70% of the sand flies were female, and of these sand flies, 8% contained visible blood. Phlebotomus alexandri Sinton, Phlebotomus papatasi Scopoli, Phlebotomus sergenti Parrot, and Sergentomyia spp. accounted for 30, 24, 1, and 45% of the sand flies that were identified, respectively. P. alexandri was more abundant earlier in the season (April and May) than P. papatasi, whereas P. papatasi predominated later in the season (August and September). Studies on the nocturnal activity of sand flies indicated that they were most active early in the evening during the cooler months, whereas they were more active in the middle of the night during the hotter months. Light traps placed inside tents with and without air conditioners collected 83 and 70% fewer sand flies, respectively, than did light traps placed outside the tents. The implications of these findings to Leishmania transmission in the vicinity of Tallil Air Base are discussed.

Illustrated keys to the mosquitoes of Thailand V. Genera Orthopodomyia, Kimia, Malaya, Topomyia, Tripteroides, and Toxorhynchites.

Illustrated keys for the identification of the fourth-instar larvae and adults of species representing six genera of mosquitoes in Thailand are presented, along with information about the geographic distribution of the species and the habitats occupied by their immature stages. Fifty-nine species belonging to genera Orthopodomyia, Kimia, Malaya, Topomyia, Tripteroides, and Toxorhynchites are recognized. Topomyia argyropalpis, To. bifurcata, To. gracilis, To. spathulirostris, To. tenuis, To. tipuliformis, and To. vijayae are included as new country records, and ten undescribed species, four in Topomyia, four in Toxorhynchites, and two in Tripteroides, are recognized.

Comparison of PCR and microscopy for the detection of asymptomatic malaria in a Plasmodium falciparum/vivax endemic area in Thailand.

OBJECTIVE: The main objective of this study was to compare the performance of nested PCR with expert microscopy as a means of detecting Plasmodium parasites during active malaria surveillance in western Thailand. METHODS: The study was performed from May 2000 to April 2002 in the village of Kong Mong Tha, located in western Thailand. Plasmodium vivax (PV) and Plasmodium falciparum (PF) are the predominant parasite species in this village, followed by Plasmodium malariae (PM) and Plasmodium ovale (PO). Each month, fingerprick blood samples were taken from each participating individual and used to prepare thick and thin blood films and for PCR analysis. RESULTS: PCR was sensitive (96%) and specific (98%) for malaria at parasite densities > or = 500/microl; however, only 18% (47/269) of P. falciparum- and 5% (20/390) of P. vivax-positive films had parasite densities this high. Performance of PCR decreased markedly at parasite densities <500/microl, with sensitivity of only 20% for P. falciparum and 24% for P. vivax at densities <100 parasites/microl. CONCLUSION: Although PCR performance appeared poor when compared to microscopy, data indicated that the discrepancy between the two methods resulted from poor performance of microscopy at low parasite densities rather than poor performance of PCR. These data are not unusual when the diagnostic method being evaluated is more sensitive than the reference method. PCR appears to be a useful method for detecting Plasmodium parasites during active malaria surveillance in Thailand.

Impact of phlebotomine sand flies on U.S. Military operations at Tallil Air Base, Iraq: 1. background, military situation, and development of a "Leishmaniasis Control Program".

One of the most significant modern day efforts to prevent and control an arthropod-borne disease during a military deployment occurred when a team of U.S. military entomologists led efforts to characterize, prevent, and control leishmaniasis at Tallil Air Base (TAB), Iraq, during Operation Iraqi Freedom. Soon after arriving at TAB on 22 March 2003, military entomologists determined that 1) high numbers of sand flies were present at TAB, 2) individual soldiers were receiving many sand fly bites in a single night, and 3) Leishmania parasites were present in 1.5% of the female sand flies as determined using a real-time (fluorogenic) Leishmania-generic polymerase chain reaction assay. The rapid determination that leishmaniasis was a specific threat in this area allowed for the establishment of a comprehensive Leishmaniasis Control Program (LCP) over 5 mo before the first case of leishmaniasis was confirmed in a U.S. soldier deployed to Iraq. The LCP had four components: 1) risk assessment, 2) enhancement of use of personal protective measures by all personnel at TAB, 3) vector and reservoir control, and 4) education of military personnel about sand flies and leishmaniasis. The establishment of the LCP at TAB before the onset of any human disease conclusively demonstrated that entomologists can play a critical role during military deployments.

Population dynamics of sporogony for Plasmodium vivax parasites from western Thailand developing within three species of colonized Anopheles mosquitoes.

BACKGROUND: The population dynamics of Plasmodium sporogony within mosquitoes consists of an early phase where parasite abundance decreases during the transition from gametocyte to oocyst, an intermediate phase where parasite abundance remains static as oocysts, and a later phase where parasite abundance increases during the release of progeny sporozoites from oocysts. Sporogonic development is complete when sporozoites invade the mosquito salivary glands. The dynamics and efficiency of this developmental sequence were determined in laboratory strains of Anopheles dirus, Anopheles minimus and Anopheles sawadwongporni mosquitoes for Plasmodium vivax parasites circulating naturally in western Thailand. METHODS: Mosquitoes were fed blood from 20 symptomatic Thai adults via membrane feeders. Absolute densities were estimated for macrogametocytes, round stages (= female gametes/zygotes), ookinetes, oocysts, haemolymph sporozoites and salivary gland sporozoites. From these census data, five aspects of population dynamics were analysed; 1) changes in life-stage prevalence during early sporogony, 2) kinetics of life-stage formation, 3) efficiency of life-stage transitions, 4) density relationships between successive life-stages, and 5) parasite aggregation patterns. RESULTS: There was no difference among the three mosquito species tested in total losses incurred by P. vivax populations during early sporogony. Averaged across all infections, parasite populations incurred a 68-fold loss in abundance, with losses of ca. 19-fold, 2-fold and 2-fold at the first (= gametogenesis/fertilization), second (= round stage transformation), and third (= ookinete migration) life-stage transitions, respectively. However, total losses varied widely among infections, ranging from 6-fold to over 2,000-fold loss. Losses during gametogenesis/fertilization accounted for most of this variability, indicating that gametocytes originating from some volunteers were more fertile than those from other volunteers. Although reasons for such variability were not determined, gametocyte fertility was not correlated with blood haematocrit, asexual parasitaemia, gametocyte density or gametocyte sex ratio. Round stages and ookinetes were present in mosquito midguts for up to 48 hours and development was asynchronous. Parasite losses during fertilization and round stage differentiation were more influenced by factors intrinsic to the parasite and/or factors in the blood, whereas ookinete losses were more strongly influenced by mosquito factors. Oocysts released sporozoites on days 12 to 14, but even by day 22 many oocysts were still present on the midgut. The per capita production was estimated to be approximately 500 sporozoites per oocyst and approximately 75% of the sporozoites released into the haemocoel successfully invaded the salivary glands. CONCLUSION: The major developmental bottleneck in early sporogony occurred during the transition from macrogametocyte to round stage. Sporozoite invasion into the salivary glands was very efficient. Information on the natural population dynamics of sporogony within malaria-endemic areas may benefit intervention strategies that target early sporogony (e.g., transmission blocking vaccines, transgenic mosquitoes).

Illustrated keys to the mosquitoes of Thailand III. Genera Aedeomyia, Ficalbia, Mimomyia, Hodgesia, Coquillettidia, Mansonia, and Uranotaenia.

Illustrated keys for the identification of fourth-instar larvae and adult females of seven genera of mosquitoes are presented, along with the geographic distribution of each species and the known immature habitats. Sixty-seven mosquito species belonging to genera Aedeomyia, Ficalbia, Mimomyia, Hodgesia, Coquillettidia, Mansonia, and Uranotaenia are recognized in Thailand. Hodgesia bailyi and Uranotaenia (Pfc.) abstrusa are included as new country records, and four new undescribed species in the subgenus Uranotaenia are reported.

Establishment of a human hepatocyte line that supports in vitro development of the exo-erythrocytic stages of the malaria parasites Plasmodium falciparum and P. vivax.

Our understanding of the biology of malaria parasite liver stages is limited because of the lack of efficient in vitro systems that support the exo-erythrocytic (EE) development of the parasite. We report the development of a new hepatocyte line (HC-04) from normal human liver cells. The HC-04 cells have proliferated in hormone-free medium for more than 200 passages. The cells were hyperdiploid, resembled liver parenchymal cells, and synthesized major liver-specific proteins and enzymes. Using Plasmodium falciparum and P. vivax sporozoites harvested from salivary glands of infected mosquitoes, we showed that HC-04 cells supported the complete EE development of these two most prevalent human malaria parasites. The EE parasites attained full maturation as shown by their infectivity to human erythrocytes. The infection rates of the liver cells were estimated to be 0.066% and 0.041% for P. falciparum and P. vivax, respectively. As the first human hepatocyte line known to support complete EE development of both P. falciparum and P. vivax, HC-04 will provide an experimental model that can be used for studying the biology of liver stage malaria parasites.