Scrub Typhus Outbreak in Chonburi Province, Central Thailand, 2013.

Investigation of a scrub typhus outbreak in Thailand during September 2013 found that 9.1% of Thai soldiers and 11.1% of residents living in areas surrounding training sites had antibodies against the causative agent, Orientia tsutsugamushi. Sequence analysis of O. tsutsugamushi from rodents and chiggers identified 7 genogroups and 3 genotypes.

Identification and Characterization of Orientia chuto in Trombiculid Chigger Mites Collected from Wild Rodents in Kenya.

We present data that concurs with the reported geographical expansion of scrub typhus outside the "Tsutsugamushi Triangle" and addition of Orientia chuto as a second species in the Orientia genus. Wild rodents were caught in Marigat, Baringo County, Kenya, and ectoparasites, including chiggers, were recovered. Rodent and chigger species were identified by taxonomic features. DNA was extracted from the chiggers and used to amplify and/or sequence the 47-kDa high temperature transmembrane protein (TSA47), the 56-kDa type-specific antigen (TSA56), and the 16S rRNA (rrs) Orientia genes. The main rodent hosts identified were Acomys wilsoni, Crocidura sp., and Mastomys natalensis, which accounted for 59.2% of the total collection. Of these, A. wilsoni and M. natalensis harbored most of the chiggers that belonged to the Neotrombicula and Microtrombicula genera. A pool of chiggers from one of M. natalensis was positive for Orientia by TSA47 PCR, but Orientia did not amplify with the TSA56 primers. On sequencing the 850 bp of the TSA47 gene, the closest phylogenetic relative was O. chuto, with 97.65% sequence homology compared to 84.63 to 84.76% for O. tsutsugamushi 16S rRNA deep sequencing also revealed O. chuto as the closest phylogenetic relative, with 99.75% sequence homology. These results and the existing immunological and molecular reports are strongly suggestive of the existence of Orientia species in Kenya.

Stabilization of RDT target antigens present in dried Plasmodium falciparum-infected samples for validating malaria rapid diagnostic tests at the point of care.

BACKGROUND: Malaria rapid diagnostic tests (RDTs) are a great achievement in implementation of parasite based diagnosis as recommended by World Health Organization. A major drawback of RDTs is lack of positive controls to validate different batches/lots at the point of care. Dried Plasmodium falciparum-infected samples with the RDT target antigens have been suggested as possible positive control but their utility in resource limited settings is hampered by rapid loss of activity over time. METHODS: This study evaluated the effectiveness of chemical additives to improve long term storage stability of RDT target antigens (HRP2, pLDH and aldolase) in dried P. falciparum-infected samples using parasitized whole blood and culture samples. Samples were treated with ten selected chemical additives mainly sucrose, trehalose, LDH stabilizer and their combinations. After baseline activity was established, the samples were air dried in bio-safety cabinet and stored at room temperatures (~ 25 °C). Testing of the stabilized samples using SD Bioline, BinaxNOW, CareStart, and First Response was done at intervals for 53 weeks. RESULTS: Stability of HRP2 at ambient temperature was reported at 21-24 weeks while that of PAN antigens (pLDH and aldolase) was 2-18 weeks of storage at all parasite densities. The ten chemical additives increased the percentage stability of HRP2 and PAN antigens. Sucrose alone and its combinations with Alsever's solution or biostab significantly increased stability of HRP2 by 56% at 2000 p/µL (p < 0.001). Trehalose and its combinations with biostab, sucrose or glycerol significantly increased stability of HRP2 by 57% (p < 0.001). Unlike sucrose, the stability of the HRP2 was significantly retained by trehalose at lower concentrations (500, and 200 p/µL). Trehalose in combination biostab stabilizer increased the percentage stability of PAN antigens by 42, and 32% at 2000 and 500 p/µL respectively (p < 0.01). This was also the chemical combination with the shortest reconstitution time (~ < 20 min). CONCLUSIONS: These findings confirm that stabilizing RDT target antigens in dried P. falciparum-infected samples using chemical additives provides field-stable positive controls for malaria RDTs.

Comparative evaluation of fluorescent in situ hybridization and Giemsa microscopy with quantitative real-time PCR technique in detecting malaria parasites in a holoendemic region of Kenya.

BACKGROUND: Early and accurate diagnosis of malaria is important in treatment as well as in the clinical evaluation of drugs and vaccines. Evaluation of Giemsa-stained smears remains the gold standard for malaria diagnosis, although diagnostic errors and potential bias estimates of protective efficacy have been reported in practice. Plasmodium genus fluorescent in situ hybridization (P-Genus FISH) is a microscopy-based method that uses fluorescent labelled oligonucleotide probes targeted to pathogen specific ribosomal RNA fragments to detect malaria parasites in whole blood. This study sought to evaluate the diagnostic performance of P-Genus FISH alongside Giemsa microscopy compared to quantitative reverse transcription polymerase chain reaction (qRT-PCR) in a clinical setting. METHOD: Five hundred study participants were recruited prospectively and screened for Plasmodium parasites by P-Genus FISH assay, and Giemsa microscopy. The microscopic methods were performed by two trained personnel and were blinded, and if the results were discordant a third reading was performed as a tie breaker. The diagnostic performance of both methods was evaluated against qRT-PCR as a more sensitive method. RESULTS: The number of Plasmodium positive cases was 26.8% by P-Genus FISH, 33.2% by Giemsa microscopy, and 51.2% by qRT-PCR. The three methods had 46.8% concordant results with 61 positive cases and 173 negative cases. Compared to qRT-PCR the sensitivity and specificity of P-Genus FISH assay was 29.3 and 75.8%, respectively, while microscopy had 58.2 and 93.0% respectively. Microscopy had a higher positive and negative predictive values (89.8 and 68.0% respectively) compared to P-Genus FISH (56.0 and 50.5%). In overall, microscopy had a good measure of agreement (76%, k = 0.51) compared to P-Genus FISH (52%, k = 0.05). CONCLUSION: The diagnostic performance of P-Genus FISH was shown to be inferior to Giemsa microscopy in the clinical samples. This hinders the possible application of the method in the field despite the many advantages of the method especially diagnosis of low parasite density infections. The P-Genus assay has great potential but application of the method in clinical setting would rely on extensive training of microscopist and continuous proficiency testing.

Evaluation of a laboratory quality assurance pilot programme for malaria diagnostics in low-transmission areas of Kenya, 2013.

BACKGROUND: One objective of the Kenya National Malaria Strategy 2009-2017 is scaling access to prompt diagnosis and effective treatment. In 2013, a quality assurance (QA) pilot was implemented to improve accuracy of malaria diagnostics at selected health facilities in low-transmission counties of Kenya. Trends in malaria diagnostic and QA indicator performance during the pilot are described. METHODS: From June to December 2013, 28 QA officers provided on-the-job training and mentoring for malaria microscopy, malaria rapid diagnostic tests and laboratory QA/quality control (QC) practices over four 1-day visits at 83 health facilities. QA officers observed and recorded laboratory conditions and practices and cross-checked blood slides for malaria parasite presence, and a portion of cross-checked slides were confirmed by reference laboratories. RESULTS: Eighty (96%) facilities completed the pilot. Among 315 personnel at pilot initiation, 13% (n = 40) reported malaria diagnostics training within the previous 12 months. Slide positivity ranged from 3 to 7%. Compared to the reference laboratory, microscopy sensitivity ranged from 53 to 96% and positive predictive value from 39 to 53% for facility staff and from 60 to 96% and 52 to 80%, respectively, for QA officers. Compared to reference, specificity ranged from 88 to 98% and negative predictive value from 98 to 99% for health-facility personnel and from 93 to 99% and 99%, respectively, for QA officers. The kappa value ranged from 0.48-0.66 for facility staff and 0.57-0.84 for QA officers compared to reference. The only significant test performance improvement observed for facility staff was for specificity from 88% (95% CI 85-90%) to 98% (95% CI 97-99%). QA/QC practices, including use of positive-control slides, internal and external slide cross-checking and recording of QA/QC activities, all increased significantly across the pilot (p < 0.001). Reference material availability also increased significantly; availability of six microscopy job aids and seven microscopy standard operating procedures increased by a mean of 32 percentage points (p < 0.001) and 38 percentage points (p < 0.001), respectively. CONCLUSIONS: Significant gains were observed in malaria QA/QC practices over the pilot. However, these advances did not translate into improved accuracy of malaria diagnostic performance perhaps because of the limited duration of the QA pilot implementation.

Factors associated with malaria microscopy diagnostic performance following a pilot quality-assurance programme in health facilities in malaria low-transmission areas of Kenya, 2014.

BACKGROUND: Malaria accounts for ~21% of outpatient visits annually in Kenya; prompt and accurate malaria diagnosis is critical to ensure proper treatment. In 2013, formal malaria microscopy refresher training for microscopists and a pilot quality-assurance (QA) programme for malaria diagnostics were independently implemented to improve malaria microscopy diagnosis in malaria low-transmission areas of Kenya. A study was conducted to identify factors associated with malaria microscopy performance in the same areas. METHODS: From March to April 2014, a cross-sectional survey was conducted in 42 public health facilities; 21 were QA-pilot facilities. In each facility, 18 malaria thick blood slides archived during January-February 2014 were selected by simple random sampling. Each malaria slide was re-examined by two expert microscopists masked to health-facility results. Expert results were used as the reference for microscopy performance measures. Logistic regression with specific random effects modelling was performed to identify factors associated with accurate malaria microscopy diagnosis. RESULTS: Of 756 malaria slides collected, 204 (27%) were read as positive by health-facility microscopists and 103 (14%) as positive by experts. Overall, 93% of slide results from QA-pilot facilities were concordant with expert reference compared to 77% in non-QA pilot facilities (p < 0.001). Recently trained microscopists in QA-pilot facilities performed better on microscopy performance measures with 97% sensitivity and 100% specificity compared to those in non-QA pilot facilities (69% sensitivity; 93% specificity; p < 0.01). The overall inter-reader agreement between QA-pilot facilities and experts was κ = 0.80 (95% CI 0.74-0.88) compared to κ = 0.35 (95% CI 0.24-0.46) between non-QA pilot facilities and experts (p < 0.001). In adjusted multivariable logistic regression analysis, recent microscopy refresher training (prevalence ratio [PR] = 13.8; 95% CI 4.6-41.4), ≥5 years of work experience (PR = 3.8; 95% CI 1.5-9.9), and pilot QA programme participation (PR = 4.3; 95% CI 1.0-11.0) were significantly associated with accurate malaria diagnosis. CONCLUSIONS: Microscopists who had recently completed refresher training and worked in a QA-pilot facility performed the best overall. The QA programme and formal microscopy refresher training should be systematically implemented together to improve parasitological diagnosis of malaria by microscopy in Kenya.

Sugar and Multivitamin Diet Effects on The Longevity and Mating Capacity of Laboratory-Reared Male Anopheline Mosquitoes.

Successful mating by male mosquitoes is dependent on several factors, with sugar feeding being particularly important. The effect of ingested vitamins on adult male mosquitoes is poorly understood. This laboratory study used 3 anopheline species, Anopheles campestris, An. dirus, and An. sawadwongporni, to study the effect of sugar and vitamins on male longevity, copulation, and fecundity. Males were fed 1 of 5 diets containing different combinations of sugar and vitamins: 10% glucose, 10% sucrose, 10% multivitamin syrup, 10% multivitamin syrup + 10% glucose, and 10% multivitamin syrup + 10% sucrose. The longevity of males was measured for a period of 15 days. Forced mating was used to simulate copulation, and fecundity was measured by counting the number of eggs oviposited and the hatch rate of larvae. The longevity of An. campestris and An. dirus was greatest when fed a diet of 10% multivitamin syrup + 10% glucose, and the longevity of An. sawadwongporni was greatest when fed a diet of 10% multivitamin syrup + 10% sucrose. The 1st mating routinely produced the most viable eggs when males were mated with several females. The diet of 10% multivitamin syrup + 10% sucrose produced numerically greater egg production and larval emergence for all 3 species, although this was not always statistically significant due to variability and small sample size. These results indicate that the addition of multivitamin syrup to sucrose may produce healthier and more fit male anophelines. This has potential implications for increasing insectary operations and improving the fitness of laboratory-reared male mosquitoes that will be released for mosquito and disease-pathogen control studies.

Field evaluation of diagnostic performance of malaria rapid diagnostic tests in western Kenya.

BACKGROUND: Malaria continues to be a major burden in the endemic regions of Kenya. Health outcomes associated with case management are dependent on the use of appropriate diagnostic methods. Rapid diagnostic tests (RDTs) have provided an important tool to help implement the WHO recommended parasite-based diagnosis in regions where expert microscopy is not available. One of the questions that must be answered when implementing RDTs is whether these tests are useful in a specific endemic region, as well as the most appropriate RDT to use. Data on the sensitivity and specificity of RDT test kits is important information to help guide test selection by national malaria control programmes. METHODS: This study evaluated the diagnostic performance of RDTs including First Response (FR), CareStart (CS), SD Bioline (SD), and Binax Now (BN). The performance of these malaria kits was compared to microscopy, the gold standard, for the detection of malaria parasites. The malaria RDTs were also compared to PCR which is a more sensitive reference test. Five-hundred participants were included in the study through community screening (50 %) and testing suspected malaria cases referred from health facilities. RESULTS: Of the 500 participants recruited, 33 % were malaria positive by microscopy while 51.2 % were positive by PCR. Compared to microscopy, the sensitivity of eight RDTs to detect malaria parasites was 90.3-94.8 %, the specificity was 73.3-79.3 %, the positive predictive value was 62.2-68.8 %, and the negative predictive value was 94.3-96.8 %. Compared to PCR, the sensitivity of the RDTs to detect malaria parasites was 71.1-75.4 %, the specificity was 80.3-84.4 %, the positive predictive value was 80.3-83.3 %, and the negative predictive value was 73.7-76.1 %. The RDTs had a moderate measure of agreement with both microscopy (>80.1 %) and PCR (>77.6 %) with a κ > 0.6. CONCLUSION: The performance of the evaluated RDTs using field samples was moderate; hence they can significantly improve the quality of malaria case management in endemic regions in Kenya by ensuring appropriate treatment of malaria positive individuals and avoiding indiscriminate use of anti-malarial drugs for parasite negative patients.

Immuno-chromatographic wicking assay for the rapid detection of dengue viral antigens in mosquitoes (Diptera: Culicidae).

There is a threat for dengue virus (DENV) reemergence in many regions of the world, particularly in areas where the DENV vectors, Aedes aegypti (L.) and Aedes albopictus (Skuse), are readily available. However, there are currently no accurate and reliable diagnostic methods to provide critical, real-time information for early detection of DENV within the vector populations to implement appropriate vector control and personal protective measures. In this article, we report the ability of an immuno-chromatographic assay developed by VecTOR Test Systems Inc. to detect DENV in a pool of female Aedes mosquitoes infected with any of the four viral serotypes. The DENV dipstick assay was simple to use, did not require a cold chain, and provided clear results within 30 min. It was highly specific and did not cross-react with samples spiked with West Nile, yellow fever, Japanese encephalitis, Rift Valley fever, chikungunya, Venezuelan equine encephalomyelitis, Ross River, LaCrosse, or Caraparu viruses. The DENV assay can provide real-time critical information on the presence of DENV in mosquitoes to public health personnel. Results from this assay will allow a rapid threat assessment and the focusing of vector control measures in high-risk areas.

Misclassification of Plasmodium infections by conventional microscopy and the impact of remedial training on the proficiency of laboratory technicians in species identification.

BACKGROUND: Malaria diagnosis is largely dependent on the demonstration of parasites in stained blood films by conventional microscopy. Accurate identification of the infecting Plasmodium species relies on detailed examination of parasite morphological characteristics, such as size, shape, pigment granules, besides the size and shape of the parasitized red blood cells and presence of cell inclusions. This work explores misclassifications of four Plasmodium species by conventional microscopy relative to the proficiency of microscopists and morphological characteristics of the parasites on Giemsa-stained blood films. CASE DESCRIPTION: Ten-day malaria microscopy remedial courses on parasite detection, species identification and parasite counting were conducted for public health and research laboratory personnel. Proficiency in species identification was assessed at the start (pre) and the end (post) of each course using known blood films of Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax infections with densities ranging from 1,000 to 30,000 parasites/µL. Outcomes were categorized as false negative, positive without speciation, P. falciparum, P. malariae, P. ovale, P. vivax and mixed infections. DISCUSSION AND EVALUATION: Reported findings are based on 1,878 P. falciparum, 483 P. malariae, 581 P. ovale and 438 P. vivax cumulative results collated from 2008 to 2010 remedial courses. Pre-training false negative and positive misclassifications without speciation were significantly lower on P. falciparum infections compared to non-falciparum infections (p < 0.0001). Post-training misclassifications decreased significantly compared to pre- training misclassifications which in turn led to significant improvements in the identification of the four species. However, P. falciparum infections were highly misclassified as mixed infections, P. ovale misclassified as P. vivax and P. vivax similarly misclassified as P. ovale (p < 0.05). CONCLUSION: These findings suggest that the misclassification of malaria species could be a common occurrence especially where non-falciparum infections are involved due to lack of requisite skills in microscopic diagnosis and variations in morphological characteristics within and between Plasmodium species. Remedial training might improve reliability of conventional light microscopy with respect to differentiation of Plasmodium infections.

Minimal Rickettsial Infection Rates and Distribution of Ticks in Uganda: An Assessment of the Seasonal Effects and Relevance to Tick-Borne Disease Risk in East Africa.

Tick-borne diseases (TBDs) pose a significant risk to humans and represent one of the major factors influencing readiness within the United States' military worldwide. Additionally, ticks and TBDs constitute major animal health problems leading to economic losses at multiple levels affecting low- and middle-income countries the hardest. Tick control is frequently hampered by issues ranging from acaricide resistance to lack of data on tick distribution and infection rates. We conducted a cross-sectional study to assess tick species distribution, host use, and rickettsial pathogen infection rate of ticks in different areas of the Uganda Cattle Corridor. We identified 4,425 hard ticks (Ixodida: Ixodidae) comprised of seven species by morphological characters with 3,315 ticks collected from four locations during the dry season and 1,110 ticks from one location during the wet season. Rickettsial pathogen prevalence was assessed in ticks collected from two districts to determine the minimum infection rate compared across seasons, village location, and tick species. We found statistically significant differences in the abundance and distribution of tick species among districts in the dry season, host animal species, and the proportion of rickettsial positive pools between villages. Seasonality, village location, and tick species do not affect the minimum infection rate of rickettsial pathogens of ticks in Uganda, but village location affects the proportion of positive tick pools. These results indicate geographical and seasonal differences among pathogen-harboring ticks contributing to our understanding of the current distribution of ticks and TBDs in Uganda.

Field evaluation of a wicking assay for the rapid detection of Rift Valley fever viral antigens in mosquitoes.

Rift Valley fever virus (RVFV) causes outbreaks of severe disease in domestic ungulates as well as humans in Africa. There is a concern that outbreaks of Rift Valley fever may continue and that this virus may spread into regions where it had not previously been detected. Surveillance and rapid detection are critical to the initiation of an effective disease control program. Here we report on the field evaluation in Kenya of the VectorTest RVFV antigen assay, modeled on the VecTest assay for West Nile virus. The dipsticks provided results in <20 min, were easy to use, and did not require a laboratory with containment facilities. Although none of the field-collected mosquitoes were infected with RVFV, the dipstick provided a clear positive result with pools of field-collected mosquitoes spiked with a single positive, irradiated (to inactivate any infectious virus) mosquito. Similarly, the dipstick was able to detect virus from pools of mosquitoes captured during the RVFV outbreak in 2007. The RVFV dipstick assay was highly specific with only a single weak false positive out of 266 pools tested (specificity > 99.6%). The RVFV assay can provide a rapid, safe, easy-to-use preliminary test to alert public health personnel to the presence of RVFV in mosquitoes in a given area. Results from this assay will allow for more rapid medical threat assessments and the focusing of vector control measures on high-risk areas.

Assessing scrub typhus and rickettsioses transmission risks in the Chiang Rai province of northern Thailand.

BACKGROUND: Scrub typhus is an important disease in the Asia-Pacific countries with increasing relevance for public health worldwide. Entomological risk assessment for scrub typhus and rickettsial disease in Phu Chi Fah village-Chiang Rai was performed to determine areas at greatest risk for disease transmission in order to increase awareness of diseases to travelers and healthcare workers. METHODS: From 2016 to 2018, rodents and chiggers were collected from 7 sites covering residential, grassland, and forest areas to determine the prevalence of pathogen of interest. The correlation between land type and vector-host-pathogen interaction system was investigated. RESULT: High prevalence of O. tsutsugamushi-infected and Rickettsia-infected chiggers was observed especially in areas with grassland and forest ecotones. Chigger and rodent species composition were negatively associated with the level of human disturbance. Increased density of rodents was responsible for a higher abundance of chigger population and increased prevalence of O. tsutsugmaushi infection in chigger in the following year. CONCLUSION: Communities in the study areas have an increased exposure risk to scrub typhus and potentially spotted fever group rickettsiosis. Travellers to this endemic area should pay more attention pathogen risks so as to avoid vector and disease exposure. Seasonal rodenticidal activity may help migitate the risk of pathogen transmission.

Low Prevalence of Leptospira Carriage in Rodents in Leptospirosis-Endemic Northeastern Thailand.

Leptospirosis is a neglected zoonotic disease affecting mostly the world's tropical regions. The rural people of northeastern Thailand suffer from a large number of leptospirosis infections, and their abundant rice fields are optimal rodent habitats. To evaluate the contribution of diversity and carriage rate of pathogenic Leptospira in rodent reservoirs to leptospirosis incidence, we surveyed rodents, between 2011 and 2012, in four provinces in northeastern Thailand with the highest incidence rates of human leptospirosis cases. We used lipL32 real-time PCR to detect pathogenic Leptospira in rodent kidneys, partial 16S rRNA gene sequencing to classify the infecting Leptospira species, and whole 16S rDNA sequencing to classify species of isolated Leptospira. Overall prevalence of Leptospira infection was 3.6% (18/495). Among infected rodents, Bandicotaindica (14.3%), Rattusexulans (3.6%), and R. rattus (3.2%) had renal carriage. We identified two pathogenic Leptospira species: L. interrogans (n = 15) and L. borgpetersenii (n = 3). In addition, an L. wolffii (LS0914U) isolate was recovered from the urine of B. indica. Leptospira infection was more prevalent in low density rodent populations, such as B. indica. In contrast, there was a lower prevalence of Leptospira infection in high density rodent populations of R. exulans and R. rattus.

Surveillance for Scrub Typhus, Rickettsial Diseases, and Leptospirosis in US and Multinational Military Training Exercise Cobra Gold Sites in Thailand.

We report findings of field surveillance for disease vectors and the prevalence of Orientia tsutsugamushi, the causative agent for scrub typhus, and other Rickettsial species that cause murine typhus and spotted fever group rickettsioses, in chigger mites and small rodents; and Leptospira in rodent kidney, urine, and environmental water samples. The study sites included various Royal Thai Army military installations and other training sites, and surrounding areas where the multinational military training exercise Cobra Gold was conducted in Thailand in 2017 and 2018. The overall prevalence of O. tsutsugamushi and Rickettsia infection in chiggers was 1.3% (20/1,594) and 7.5% (119/1,594), respectively. Serum samples of the captured rodents indicated previous exposure to O. tsutsugamushi infection with a seropositive rate of 12.2%. Leptospira species were isolated from rodent kidneys and water samples collected from catchment areas as well as tap water used for hand washing. Findings from this surveillance are important in determining the potential for scrub typhus, rickettsioses, and leptospirosis risk to military and US government personnel, as well as for informing regional and combatant commanders for prevention, correct diagnosis, prompt treatment, and timely and focused implementation of vector control and personal protective measures.

Colonization and Maintenance of Anopheles belenrae and Anopheles pullus From the Republic Of Korea.

The Anopheles Hyrcanus Group in the Republic of Korea (ROK) consists of 5 morphologically indistinct species that can only be identified with certainty by polymerase chain reaction (PCR). A total of 86 bloodfed Anopheles spp. were collected from a cow barn located in the village of Tongilchon near the demilitarized zone in the ROK on June 13, 2016, and sent to the Armed Forces Research Institute of Medical Sciences in Bangkok, Thailand, where they were identified to species by PCR. The 1st shipment contained 15 An. belenrae and 37 An. pullus females that were used to start the colonies. Parent females that oviposited were identified by PCR for colonization. A higher proportion of F1-F4 females of An. belenrae than An. pullus bloodfed when provided both blood meals on human arms and using a membrane feeding system with human blood. Following blood meals, the females were forced mated for colony maintenance. The mean numbers of eggs oviposited per female for An. belenrae was 127.7 ± 19.3 and for An. pullus was 136 ± 23.6. On average, at 25°C (±2°C) An. belenrae and An. pullus took 15.1 and 16.1 days to develop from egg to adult, respectively. A 2nd group of bloodfed Anopheles spp. was collected at the same location in the ROK on June 24, 2017. This group contained 13 An. belenrae and 27 An. pullus. Similarly, eggs were obtained and adults identified by PCR and then reared to adults and subsequent generations forced mated to members of each of the existing colonies to increase genetic diversity. The colonies were established to evaluate their susceptibility to vector vivax malaria, which is essential to better understand the epidemiology of malaria transmission in Korea. This is the 1st report of colonization of both An. belenrae and An. pullus.

Importance of algal biomass to growth and development of Anopheles gambiae larvae.

We conducted experiments to investigate the importance of algal food resources for larval growth and adult emergence of Anopheles gambiae Giles s.s. in simulated larval habitats in Kenya, and in greenhouse and laboratory microcosms in the United States. In the first experiment, we used shading to reduce algal biomass, and because algal production and larval development might be a function of underlying soil nutrients, we crossed sun-shade treatments with soils of two distinct types collected near larval habitats. Shading reduced pupation rates and total adult biomass of An. gambiae by approximately 50%. Soil type had no significant effect on mosquito production, but it did significantly affect concentrations of phosphorus and chlorophyll a in the surface microlayer. In a subsequent experiment conducted in the greenhouse to reduce temperature differences found between the shaded and sunlit treatments, <1% of larvae in the shaded treatments reached the pupal stage. There was a marked reduction of chlorophyll a levels as a function of shading and larval density. In a third experiment, larvae receiving material harvested from sunlit surface microlayers performed as well as those receiving liver powder, whereas those receiving surface microlayer from shaded habitats suffered >90% mortality and failed to pupate. In a fourth experiment, glucose was added to shaded microcosms to stimulate bacterial activity in the absence of algae. Bacterial growth rates were 2 to 3 times higher, and larval development was enhanced in glucose-amended treatments. However, pupation rates and adult weights in glucose-amended shaded microcosms were still poor compared with those in nonamended sunlit microcosms. Overall, these results demonstrate the importance of algal biomass in the surface microlayers of larval habitats to development and adult production of An. gambiae.

Comparative efficacy of existing surveillance tools for Aedes aegypti in Western Kenya.

All traditional surveillance techniques for Aedes aegypti have been developed for the cosmopolitan domestic subspecies Ae. aegypti aegypti, and not the sylvatic subspecies, Ae. aegypti formosus. The predominant form in Western Kenya is Ae. aegypti formosus that is rarely associated with human habitations but is linked to transmission of sylvatic dengue virus strains. We compared five surveillance methods for their effectiveness in sampling Ae. aegypti formosus with the goal of determining a sustainable surveillance strategy in Kenya. The methods included larval and pupal surveys, oviposition trapping, BG-Sentinel trapping, resting boxes, and backpack aspirations. Larval and pupal surveys collected the highest number of Ae. aegypti formosus (51.3%), followed by oviposition traps (45.7%), BG-Sentinel traps (3.0%), and zero collected with either backpack aspiration or resting box collections. No Ae. aegypti formosus larvae or pupae were found indoors. The results indicate that oviposition traps and outdoor larval and pupal surveys were better surveillance methods for Ae. aegypti formosus in Western Kenya.

Characterization of Plasmodium ovale curtisi and P. ovale wallikeri in Western Kenya utilizing a novel species-specific real-time PCR assay.

BACKGROUND: Plasmodium ovale is comprised of two genetically distinct subspecies, P. ovale curtisi and P. ovale wallikeri. Although P. ovale subspecies are similar based on morphology and geographical distribution, allelic differences indicate that P. ovale curtisi and P. ovale wallikeri are genetically divergent. Additionally, potential clinical and latency duration differences between P. ovale curtisi and P. ovale wallikeri demonstrate the need for investigation into the contribution of this neglected malaria parasite to the global malaria burden. METHODS: In order to detect all P. ovale subspecies simultaneously, we developed an inclusive P. ovale-specific real-time PCR assay based on conserved regions between P. ovale curtisi and P. ovale wallikeri in the reticulocyte binding protein 2 (rbp2) gene. Additionally, we characterized the P. ovale subspecies prevalence from 22 asymptomatic malaria infections using multilocus genotyping to discriminate P. ovale curtisi and P. ovale wallikeri. RESULTS: Our P. ovale rbp2 qPCR assay validation experiments demonstrated a linear dynamic range from 6.25 rbp2 plasmid copies/microliter to 100,000 rbp2 plasmid copies/microliter and a limit of detection of 1.5 rbp2 plasmid copies/microliter. Specificity experiments showed the ability of the rbp2 qPCR assay to detect low-levels of P. ovale in the presence of additional malaria parasite species, including P. falciparum, P. vivax, and P. malariae. We identified P. ovale curtisi and P. ovale wallikeri in Western Kenya by DNA sequencing of the tryptophan-rich antigen gene, the small subunit ribosomal RNA gene, and the rbp2 gene. CONCLUSIONS: Our novel P. ovale rbp2 qPCR assay detects P. ovale curtisi and P. ovale wallikeri simultaneously and can be utilized to characterize the prevalence, distribution, and burden of P. ovale in malaria endemic regions. Using multilocus genotyping, we also provided the first description of the prevalence of P. ovale curtisi and P. ovale wallikeri in Western Kenya, a region holoendemic for malaria transmission.

The role of Pfmdr1 and Pfcrt in changing chloroquine, amodiaquine, mefloquine and lumefantrine susceptibility in western-Kenya P. falciparum samples during 2008-2011.

Single Nucleotide Polymorphisms (SNPs) in the Pfmdr1, and Pfcrt, genes of Plasmodium falciparum may confer resistance to a number of anti-malaria drugs. Pfmdr1 86Y and haplotypes at Pfcrt 72-76 have been linked to chloroquine (CQ) as well as amodiaquine (AQ) resistance. mefloquine (MQ) and lumefantrine (LU) sensitivities are linked to Pfmdr1 86Y. Additionally, Pfcrt K76 allele carrying parasites have shown tolerance to LU. We investigated the association between Pfmdr1 86/Pfcrt 72-76 and P. falciparum resistance to CQ, AQ, MQ and LU using field samples collected during 2008-2011 from malaria endemic sites in western Kenya. Genomic DNA from these samples was genotyped to examine SNPs and haplotypes in Pfmdr1 and Pfcrt respectively. Additionally, immediate ex vivo and in vitro drug sensitivity profiles were assessed using the malaria SYBR Green I fluorescence-based assay. We observed a rapid but steady percent increase in wild-type parasites with regard to both Pfmdr1 and Pfcrt between 2008 and 2011 (p<0.0001). Equally, a significant reciprocate decrease in AQ and CQ median IC50 values occurred (p<0.0001) during the same period. Thus, the data in this study point to a significantly rapid change in parasite response to AQ and CQ in the study period. This may be due to releasing of drug pressure on the parasite from reduced use of AQ in the face of increased Artemisinin (ART) Combination Therapy (ACT) administration following the intervention of the Global Fund in 2008. LU has been shown to select for 76K genotypes, thus the observed increase in 76K genotypes coupled with significant cross resistance between LU and MQ, may herald emergence of tolerance against both drugs in future.