Daibin Zhong

Changing Patterns of Malaria Epidemiology between 2002 and 2010 in Western Kenya: The Fall and Rise of Malaria

Background The impact of insecticide treated nets (ITNs) on reducing malaria incidence is shown mainly through data collection from health facilities. Routine evaluation of long-term epidemiological and entomological dynamics is currently unavailable. In Kenya, new policies supporting the provision of free ITNs were implemented nationwide in June 2006. To evaluate the impacts of ITNs on malaria transmission, we conducted monthly surveys in three sentinel sites with different transmission intensities in western Kenya from 2002 to 2010. Methods and Findings Longitudinal samplings of malaria parasite prevalence in asymptomatic school children and vector abundance in randomly selected houses were undertaken monthly from February 2002. ITN ownership and usage surveys were conducted annually from 2004 to 2010. Asymptomatic malaria parasite prevalence and vector abundances gradually decreased in all three sites from 2002 to 2006, and parasite prevalence reached its lowest level from late 2006 to early 2007. The abundance of the major malaria vectors, Anopheles funestus and An. gambiae, increased about 5–10 folds in all study sites after 2007. However, the resurgence of vectors was highly variable between sites and species. By 2010, asymptomatic parasite prevalence in Kombewa had resurged to levels recorded in 2004/2005, but the resurgence was smaller in magnitude in the other sites. Household ITN ownership was at 50–70% in 2009, but the functional and effective bed net coverage in the population was estimated at 40.3%, 49.4% and 28.2% in 2010 in Iguhu, Kombewa, and Marani, respectively. Conclusion The resurgence in parasite prevalence and malaria vectors has been observed in two out of three sentinel sites in western Kenya despite a high ownership of ITNs. The likely factors contributing to malaria resurgence include reduced efficacy of ITNs, insecticide resistance in mosquitoes and lack of proper use of ITNs. These factors should be targeted to avoid further resurgence of malaria transmission.

High prevalence of asymptomatic plasmodium falciparum infections in a highland area of western Kenya: a cohort study.

BACKGROUND Transmission of malaria in an area of hypoendemicity in the highlands of western Kenya is not expected to lead to rapid acquisition of immunity to malaria. However, the subpopulation of individuals with asymptomatic Plasmodium falciparum infection may play a significant role as an infection reservoir and should be considered in malaria-control programs. Determination of the spatiotemporal dynamics of asymptomatic subpopulations provides an opportunity to estimate the epidemiological importance of this group to malaria transmission. METHODS Monthly parasitological surveys were undertaken for a cohort of 246 schoolchildren over 12 months. The prevalence of P. falciparum infection among 2,611 blood samples was analyzed by both microscopy and polymerase chain reaction, and infection durations were determined. RESULTS Infection prevalence and duration (range, 1-12 months) decreased with age and altitude. The prevalence was high among pooled blood samples recovered from children aged 5-9 years (34.4%) and from those aged 10-14 years (34.1%) but was significantly lower among blood samples obtained from older children (9.1%). The prevalence decreased from 52.4% among pooled blood samples from children living at an altitude of approximately 1,430 m to 23.3% among pooled samples from children living at an altitude of 1,580 m. CONCLUSIONS The prevalence of asymptomatic P. falciparum infection was high, with polymerase chain reaction analysis detecting a significantly greater number of infections, compared with microscopy. Our results are consistent with gradual acquisition of immunity with increasing age upon repeated infection, and they also show that the risk of malaria transmission is highly heterogeneous in the highland area. The results provide strong support for targeted malaria-control interventions.

Comparative Transcriptome Analyses of Deltamethrin-Resistant and -Susceptible Anopheles gambiae Mosquitoes from Kenya by RNA-Seq

Malaria causes more than 300 million clinical cases and 665,000 deaths each year, and the majority of the mortality and morbidity occurs in sub-Saharan Africa. Due to the lack of effective vaccines and wide-spread resistance to antimalarial drugs, mosquito control is the primary method of malaria prevention and control. Currently, malaria vector control relies on the use of insecticides, primarily pyrethroids. The extensive use of insecticides has imposed strong selection pressures for resistance in the mosquito populations. Consequently, resistance to pyrethroids in Anopheles gambiae, the main malaria vector in sub-Saharan Africa, has become a major obstacle for malaria control. A key element of resistance management is the identification of resistance mechanisms and subsequent development of reliable resistance monitoring tools. Field-derived An. gambiae from Western Kenya were phenotyped as deltamethrin-resistant or -susceptible by the standard WHO tube test, and their expression profile compared by RNA-seq. Based on the current annotation of the An. gambiae genome, a total of 1,093 transcripts were detected as significantly differentially accumulated between deltamethrin-resistant and -susceptible mosquitoes. These transcripts are distributed over the entire genome, with a large number mapping in QTLs previously linked to pyrethorid resistance, and correspond to heat-shock proteins, metabolic and transport functions, signal transduction activities, cytoskeleton and others. The detected differences in transcript accumulation levels between resistant and susceptible mosquitoes reflect transcripts directly or indirectly correlated with pyrethroid resistance. RNA-seq data also were used to perform a de-novo Cufflinks assembly of the An. gambiae genome.

Molecular Ecology of Pyrethroid Knockdown Resistance in Culex pipiens pallens Mosquitoes

Pyrethroid insecticides have been extensively used in China and worldwide for public health pest control. Accurate resistance monitoring is essential to guide the rational use of insecticides and resistance management. Here we examined the nucleotide diversity of the para-sodium channel gene, which confers knockdown resistance (kdr) in Culex pipiens pallens mosquitoes in China. The sequence analysis of the para-sodium channel gene identified L1014F and L1014S mutations. We developed and validated allele-specific PCR and the real-time TaqMan methods for resistance diagnosis. The real-time TaqMan method is more superior to the allele-specific PCR method as evidenced by higher amplification rate and better sensitivity and specificity. Significant positive correlation between kdr allele frequency and bioassay-based resistance phenotype demonstrates that the frequency of L1014F and L1014S mutations in the kdr gene can be used as a molecular marker for deltamethrin resistance monitoring in natural Cx. pipiens pallens populations in the East China region. The laboratory selection experiment found that L1014F mutation frequency, but not L1014S mutation, responded to deltamethrin selection, suggesting that the L1014F mutation is the key mutation conferring resistance to deltamethrin. High L1014F mutation frequency detected in six populations of Cx. pipens pallens suggests high prevalence of pyrethroid resistance in Eastern China, calling for further surveys to map the resistance in China and for investigating alternative mosquito control strategies.

Genetic analysis of invasive Aedes albopictus populations in Los Angeles County, California and its potential public health impact.

The Asian tiger mosquito, Aedes albopictus, is an anthropophilic aggressive daytime-biting nuisance and an efficient vector of certain arboviruses and filarial nematodes. Over the last 30 years, this species has spread rapidly through human travel and commerce from its native tropical forests of Asia to every continent except Antarctica. In 2011, a population of Asian tiger mosquito (Aedes albopictus) was discovered in Los Angeles (LA) County, California. To determine the probable origin of this invasive species, the genetic structure of the population was compared against 11 populations from the United States and abroad, as well as preserved specimens from a 2001 introduction into California using the mitochondrial cytochrome c oxidase 1 (CO1) gene. A total of 66 haplotypes were detected among samples and were divided into three main groups. Aedes albopictus collected in 2001 and 2011 from LA County were genetically related and similar to those from Asia but distinct from those collected in the eastern and southeastern United States. In view of the high genetic similarities between the 2001 and 2011 LA samples, it is possible that the 2011 population represents in part the descendants of the 2001 introduction. There remains an imperative need for improved surveillance and control strategies for this species.

Molecular analysis of chloroquine resistance in Plasmodium falciparum in Yunnan Province, China

Resistance of Plasmodium falciparum to chloroquine (CQ) is determined by the mutation at K76T of the P. falciparum chloroquine resistance transporter (pfcrt) gene and modified by other mutations in this gene and in the P. falciparum multidrug resistance 1 (pfmdr1) gene. To determine the extent of polymorphisms in these genes in field P. falciparum isolates from Yunnan province of China, we genotyped the pfcrt codon 76, pfmdr1 codons 86 and 1246. Our results showed that although CQ has been withdrawn from treating falciparum malaria for over two decades, 90.3% of the parasites still carried the pfcrt K76T mutation. In contrast, mutations at pfmdr1 codons 86 and 1246 were rare. Sequencing analysis of the pfcrt gene in 34 parasite field isolates revealed CVIET at positions 72–76 as the major type, consistent with the theory of Southeast Asian origin of CQ resistance in the parasite. In addition, two novel pfcrt haplotypes (75D/144Y/220A and 75E/144Y/220A) were identified. Real‐time polymerase chain reaction was used to determine pfmdr1 gene amplification, which is associated with mefloquine resistance. Our result indicated that in agreement with that mefloquine has not been used in this area, most (>90%) of the parasites had one pfmdr1 copy. Genotyping at two hypervariable loci showed relatively low levels of genetic diversity of the parasite population. Meanwhile, 28.4% of cases were found to contain mixed clones, which favour genetic recombination. Furthermore, despite a unique history of antimalarial drugs in Yunnan, its geographical connections with three malarious countries facilitate gene flow among parasite populations and evolution of novel drug‐resistant genotypes. Therefore, continuous surveillance of drug resistance in this area is necessary for timely adjustment of local drug policies and more effective malaria control.

Quantitative Trait Loci Controlling Refractoriness to Plasmodium falciparum in Natural Anopheles gambiae Mosquitoes From a Malaria-Endemic Region in Western Kenya

Natural anopheline populations exhibit much variation in ability to support malaria parasite development, but the genetic mechanisms underlying this variation are not clear. Previous studies in Mali, West Africa, identified two quantitative trait loci (QTL) in Anopheles gambiae mosquitoes that confer refractoriness (failure of oocyst development in mosquito midguts) to natural Plasmodium falciparum parasites. We hypothesize that new QTL may be involved in mosquito refractoriness to malaria parasites and that the frequency of natural refractoriness genotypes may be higher in the basin region of Lake Victoria, East Africa, where malaria transmission intensity and parasite genetic diversity are among the highest in the world. Using field-derived F2 isofemale families and microsatellite marker genotyping, two loci significantly affecting oocyst density were identified: one on chromosome 2 between markers AG2H135 and AG2H603 and the second on chromosome 3 near marker AG3H93. The first locus was detected in three of the five isofemale families studied and colocalized to the same region as Pen3 and pfin1 described in other studies. The second locus was detected in two of the five isofemale families, and it appears to be a new QTL. QTL on chromosome 2 showed significant additive effects while those on chromosome 3 exhibited significant dominant effects. Identification of P. falciparum-refractoriness QTL in natural An. gambiae mosquitoes is critical to the identification of the genes involved in malaria parasite transmission in nature and for understanding the coevolution between malaria parasites and mosquito vectors.

Relationship between Knockdown Resistance, Metabolic Detoxification and Organismal Resistance to Pyrethroids in Anopheles sinensis

Anopheles sinensis is the most important vector of malaria in Southeast Asia, including China. Currently, the most effective measure to prevent malaria transmission relies on vector control through the use of insecticides, primarily pyrethroids. Extensive use of insecticides poses strong selection pressure on mosquito populations for resistance. Resistance to insecticides can arise due to mutations in the insecticide target site (target site resistance), which in the case of pyrethroids is the para-type sodium channel gene, and/or the catabolism of the insecticide by detoxification enzymes before it reaches its target (metabolic detoxification resistance). In this study, we examined deltamethrin resistance in An. sinensis from China and investigated the relative importance of target site versus metabolic detoxification mechanisms in resistance. A high frequency (>85%) of nonsynonymous mutations in the para gene was found in populations from central China, but not in populations from southern China. Metabolic detoxification as measured by the activity of monooxygenases and glutathione S-transferases (GSTs) was detected in populations from both central and southern China. Monooxygenase activity levels were significantly higher in the resistant than the susceptible mosquitoes, independently of their geographic origin. Stepwise multiple regression analyses in mosquito populations from central China found that both knockdown resistance (kdr) mutations and monooxygenase activity were significantly associated with deltamethrin resistance, with monooxygenase activity playing a stronger role. These results demonstrate the importance of metabolic detoxification in pyrethroid resistance in An. sinensis, and suggest that different mechanisms of resistance could evolve in geographically different populations.

Multiple Resistances and Complex Mechanisms of Anopheles sinensis Mosquito: A Major Obstacle to Mosquito-Borne Diseases Control and Elimination in China

Malaria, dengue fever, and filariasis are three of the most common mosquito-borne diseases worldwide. Malaria and lymphatic filariasis can occur as concomitant human infections while also sharing common mosquito vectors. The overall prevalence and health significance of malaria and filariasis have made them top priorities for global elimination and control programmes. Pyrethroid resistance in anopheline mosquito vectors represents a highly significant problem to malaria control worldwide. Several methods have been proposed to mitigate insecticide resistance, including rotational use of insecticides with different modes of action. Anopheles sinensis, an important malaria and filariasis vector in Southeast Asia, represents an interesting mosquito species for examining the consequences of long-term insecticide rotation use on resistance. We examined insecticide resistance in two An. Sinensis populations from central and southern China against pyrethroids, organochlorines, organophosphates, and carbamates, which are the major classes of insecticides recommended for indoor residual spray. We found that the mosquito populations were highly resistant to the four classes of insecticides. High frequency of kdr mutation was revealed in the central population, whereas no kdr mutation was detected in the southern population. The frequency of G119S mutation in the ace-1 gene was moderate in both populations. The classification and regression trees (CART) statistical analysis found that metabolic detoxification was the most important resistance mechanism, whereas target site insensitivity of L1014 kdr mutation played a less important role. Our results indicate that metabolic detoxification was the dominant mechanism of resistance compared to target site insensitivity, and suggests that long-term rotational use of various insecticides has led An. sinensis to evolve a high insecticide resistance. This study highlights the complex network of mechanisms conferring multiple resistances to chemical insecticides in mosquito vectors and it has important implication for designing and implementing vector resistance management strategies.

Molecular epidemiology of drug-resistant malaria in western Kenya highlands

BackgroundSince the late 1980s a series of malaria epidemics has occurred in western Kenya highlands. Among the possible factors that may contribute to the highland malaria epidemics, parasite resistance to antimalarials has not been well investigated.MethodsUsing parasites from highland and lowland areas of western Kenya, we examined key mutations associated with Plasmodium falciparum resistance to sulfadoxine – pyrimethamine and chloroquine, including dihydrofolate reductase (pfdhfr) and dihydropteroate synthetase (pfdhps), chloroquine resistance transporter gene (pfcrt), and multi-drug resistance gene 1 (pfmdr1).ResultsWe found that >70% of samples harbored 76T pfcrt mutations and over 80% of samples harbored quintuple mutations (51I/59R/108N pfdhfr and 437G/540E pfdhps) in both highland and lowland samples. Further, we did not detect significant difference in the frequencies of these mutations between symptomatic and asymptomatic malaria volunteers, and between highland and lowland samples.ConclusionThese findings suggest that drug resistance of malaria parasites in the highlands could be contributed by the mutations and their high frequencies as found in the lowland. The results are discussed in terms of the role of drug resistance as a driving force for malaria outbreaks in the highlands.