Although poor maternal mental health is a major public health problem, with detrimental effects on the individual, her children and society, information on its correlates in low-income countries is sparse.This study investigates the prevalence of common mental disorders (CMD) among at-risk mothers, and explores its associations with sociodemographic factors.This population-based survey of mothers of children aged 0–36 months used the 14-item Shona Symptom Questionnaire (SSQ). Mothers whose response was “yes” to 8 or more items on the scale were defined as “at risk of CMD.”Of the 1,922 mothers (15–48 years), 28.8% were at risk of CMD. Risk of CMD was associated with verbal abuse, physical abuse, a partner who did not help with the care of the child, being in a polygamous relationship, a partner with low levels of education, and a partner who smoked cigarettes. Cohabiting appeared to be protective.Taken together, our results indicate the significance of the quality of relations with one’s partner in shaping maternal mental health. The high proportion of mothers who are at risk of CMD emphasizes the importance of developing evidence-based mental health programmes as part of the care package aimed at improving maternal well-being in Tanzania and other similar settings. Common mental disorders (CMD), including depression, anxiety and somatic symptoms, are reported to occur with more frequency among women than men, especially in low- and low-middle income countries (LLMICs) . Poor maReports from population-based studies show the prevalence of CMD among mothers as ranging from 10 to 56% –10. The Reported sociodemographic risk factors of CMD among mothers in LLMICs include being illiterate, having an unsupportive partner, physical violence, verbal abuse, alcohol use, polygamous marriage, having at least 3 children, HIV/AIDS and the death of a loved one ,11–15.However, there have been contrasting reports on the association between some demographic risk factors and CMD. While in some studies, mothers with CMD were more likely to be older ,10,16 orThe Shona Symptom Questionnaire (SSQ) is a screening instrument for CMD which was developed and validated in Zimbabwe . In contThis report is part of a community-based cross-sectional study that was conducted in Kilimanjaro, Tanzania. The main objective of the primary study was to document the development and the factors associated with poor development of children aged 0-36 months. The main aim of this report is to document the prevalence and factors associated with the risk of common mental disorders in mothers of children aged 0-36 months.Prior to study commencement, the protocol was approved by the Kilimanjaro Christian Medical Centre Ethical Committee, the Tanzanian National Institute of Medical Research (NIMR) and the Regional Ethics Committee, South East Norway. Before joining the study, caregivers were informed about the study aims and procedures involved. Written consent was sought from the caregivers who wanted to join the study, and they were advised that they had the right to refuse to participate. In the case of illiterate caregivers, the right thumbprint was used as a signature.From June 2010 to May 2011, the study team visited 50 villages/streets within 7 districts in Kilimanjaro region, Northern Tanzania. Kilimanjaro is projected to have a population of 1,702,207, with 451,911 females aged 15–49 years . ApproxiIn principle, mental health is integrated into primary healthcare in Tanzania. At the community level, mental health-care is mainly provided by trained primary healthcare workers who are supervised by district mental health coordinators. However, this system faces a number of challenges including a shortage of skilled mental healthcare workers, insufficient funds for supervision, and a limited supply of essential medicines ,28.Mothers of children aged 0-36 months were recruited for this study. There were no specific exclusion criteria.th cluster was selected. In each cluster, a compact segment sampling method was used to select 50 children aged 0-36 months. The selected clusters (i.e. villages/streets) were mapped into segments, each with approximately 50 children aged 0-36 months (from the 2009 projections of the 2002 census). All segments were assigned a number on pieces of paper, and one was randomly picked. Within the selected segment, a quota sampling method was used to determine the children to be included in the study. The study team visited all the households until 50 children whose parents consented and who fulfilled the gender and sex criteria were examined. If all the households in the segment had been surveyed and fewer than 50 children were available, a second segment was randomly selected. The members of the households in the selected segments were informed to be available on the day of the survey. The required sample size was 2,500 children, with approximately 100-200 children in each of the 12 specified age groups.To obtain a representative sample of children aged 0-36 months for the original study, a multistage sampling design was used. First, a random cluster sampling method, with probability proportional to the size of the population, was used to select 50 clusters i.e. villages/streets within the 7 districts. This was done by listing the population of children aged 0-36 months for each village/street, with a column showing its cumulative population, to be used as a sampling frame. The village and street by single-age population data were obtained from the 2002 census (with 2009 projections), from the Kilimanjaro regional bureau of statistics office in April 2009. The sampling interval was then calculated: the total population of 0-36-month-olds was divided by the required number of clusters i.e. 166,808/50. A random number between 0 and 1 was generated from a computer, and the starting point for selection of the first cluster was determined by multiplying the random number with the sampling interval. The subsequent cluster was located by adding the sample interval to the previous number, until the 50The mothers’ mental health was screened using SSQ, a screening instrument designed to be used by lay interviewers for the measurement of non-psychotic psychological morbidities, including depressive, generalized anxiety and somatic disorders. The 14-item SSQ was developed using items from the WHO Self-Report Questionnaire, and 47 local idioms of distress of mental disorder that were identified by qualitative interviews of 110 patients with obvious psychiatric morbidities. The final list of 14 items of SSQ are made up of 7 items found in the SRQ , and 7 items locally identified and found to be relevant defining distress in the cultural context The validation of the SSQ was done using two validation criteria: (i) Revised Clinical Interview Schedule and (ii) Clinical diagnosis of mental problems by the care providers among patients attending primary health care clinics and traditional medical practitioners. Using a cut-off score of 8 or more as diagnosis of mental disorder, it had a reliable internal consistency , and the sensitivity and specificity of SSQ was 67% and 83% respectively . . The SIn the current study, the SSQ was translated into Swahili, back-translated, field-tested and revised accordingly. It was administered by the research assistants who consisted of two nurse midwives, a physiotherapist and a nutritionist, all of whom underwent training for two weeks. Mothers who scored above the cut-off of 8 were classified as ‘at risk’ and were referred for counselling and psychiatric assessment through the local health facility.The factorial structure of the SSQ in this study was investigated using a principal component analysis. The scale has a strong first factor with an eigenvalue of 5.048, explaining 36.06% of the variance. All the items on the scale strongly loaded on this factor, with factor loadings ranging from 0.72 to 0.34. The unidimensionality of the scale was further confirmed by the high internal consistency .The following information was obtained from the mothers using a structured interview guide: Maternal Information: age in years ; marital status ; whether she was in a polygamous relationship; education level ; employment status ; residence ; parity i.e. number of children ever borne ; if children were from the same partner; death of a child; whether she was verbally or physically abused by her partner in the past one month. The partner’s age in years; education level ; occupation ; alcohol and cigarette use were also recorded. The mothers who used alcohol both during pregnancy and during the study period were considered as ‘alcohol users’. Additionally, mothers were asked if their partner ever helped with taking care of the children e.g. changing clothes, bathing or feeding the child. Questions were asked about the most recent partner i.e. the father of the child in the study.2 tests. A p value was considered to be statistically significant when it was less than 0.05. The factors which were statistically significant in the univariate analysis were entered into the multivariate logistic regression model to determine significant factors associated with risk of CMD. The Odds Ratio (OR) and their 95% confidence interval (CI) were used to measure the strength of association between potential factors and risk of CMD.Double data entry was done using EpiData version 3.1. Data checking, cleaning, recoding, labelling and analysis were done using PASW18 and Stata/IC 11.0. Frequency tables and univariate descriptive analysis were carried out to investigate the spread of scores. Bivariate analyses of those ‘at risk of CMD’ and possible associated factors were carried out using cross tabulations and χp <0.001). Among the respondents, 2,354 (95.9%) were mothers, while the rest were caregivers. Mothers from one district  were excluded from this analysis because questions were added after collecting data in that area. The characteristics of these mothers differed significantly from the remaining mothers. In addition, 9 (0.4%) mothers were excluded because more than half of their information was missing.Of the 2,774 households that were approached, 2,454 (88.5%) consented to participate in the study. Compared to participants, the non-participants were more likely to reside in urban areas  and (7.3 + 2.0 v. 8.0 + 3.2 years) respectively. The sociodemographic data are summarized in Of the mothers  included in this analysis, 1,456 (75.8%) were from rural areas. Their mean age and years of education were lower than their partners’  2.74, p = 0.098).Overall, 553 (28.8%) mothers scored 8 or more on the scale, and hence were considered to be at risk of CMD. The least-reported symptoms were suicidal ideation and hallucination. An initial bivariate analysis  and 3 shAfter controlling for confounding factors  and 3, vThe results of this study suggest that more than 1 in 4 mothers are at risk of CMD. The prevalence of mothers at risk of CMD reported here is consistent with reports from other LLMICs –11,16,29Consistent with several other reports, in this study intimate partner verbal and physical abuse also independently predicted risk of CMD ,11,30,31Evidence suggests that alcohol use by either or both partners increases the severity of intimate partner violence ,32–34. HBeing married as opposed to being single was independently associated with risk of CMD in Indian women . This isEducation provides the means to cope with difficulties and to extricate oneself from poverty. Although the association between risk of CMD and the mother’s low level of education was not consistent in this study, the partner’s low level of education predicted CMD in the mother. This is consistent with a study which showed mothers with uneducated husbands to have a higher risk of persistent depression even beyond the postpartum period . In resuMothers in this study with partners who helped with child care were less likely to be at risk of CMD. In one study, mothers who got daily support with child care from at least one family member were less likely to have depression . These rHIV-infected persons with symptoms of depression were reported to have an increased risk of disease progression and poorer quality of life ,38. The The current study used the SSQ to evaluate the existence of the CMD in a large population-based study. The use of SSQ is a potential limitation since it is too brief to provide any diagnosis; however, since our aim was to screen and not diagnose, we do not find the brevity of SSQ to compromise the validity of our findings. Additionally, in the present state, the SSQ is uniquely placed to perform well in a rural Tanzanian setting, since it was developed using emic and etic approaches to suit the African cultural setting when discussing mental health issues and its simplicity allows for its use by staff that may be of relatively limited educational background.Among the limitations of this study is the refusal rate of working mothers in the urban areas to participate in the study. This could have influenced the results, as there might have been fewer mothers with higher levels of education. The verbal diagnosis of HIV used in this study could have resulted in an underestimation of the number of HIV-infected mothers. Furthermore, this was a cross-sectional study, and hence it is difficult to comment on the direction of association between partner abuse and risk of CMD. Earlier reports on the mental health effects of intimate partner violence suggest that CMD results from intimate partner violence ,41,42. OIn conclusion, the risk of CMD among mothers in this population is high. Results from this study emphasize the association between partner abuse, low levels of education, and lack of partner support in child care with risk of CMD. A culture which encourages partners to be involved in child-caring activities might also contribute to decreasing the risk of maternal CMD. A longitudinal study to establish the local risk factors for CMD would provide the basis for creating a screening tool for community use. In addition, primary healthcare providers should be provided with skills and tools to detect and manage CMD in the community.The risk of CMD among mothers of young children in our study area is a source of concern, as it is likely to lead to adverse effects on child and maternal health. There is a need to invest resources with the aim of fully understanding risk factors for CMD and developing evidence-based mental health services in order to enhance maternal mental health.


Hyperparasitaemia is a feature of childhood severe malaria but there is little information on the risk factors for hyperparasitaemia in malarious childrenPlasmodium falciparum hyperparasitaemia, defined as asexual parasitaemia > 250,000/μl, at presentation were evaluated in 3338 malarious children enrolled prospectively between 2008 and 2010 in an endemic area of southwestern Nigeria.The risk factors associated with At enrolment, 97 (3%) of 3338 malarious children had hyperparasitaemia. In a multiple regression model, 3 factors were found to be independent risk factors for the presence of hyperparasitaemia at enrolment: an age ≤ 11 years , fever , and enrolment after year 2008 . Duration of illness ≤ 3 d was associated with increased risk of hyperparasitaemia. There was no association between season and hyperparasitaemia. Compared to non-hyperparasitaemia, hyperparasitaemia was associated with an increased risk of progression to cerebral malaria (P < 0.0001). The risk of progression in hyperparasitaemic children was higher in < 5-year olds (P = 0.02).Young age and presence of fever are independent risk factors for hyperparasitaemia which is associated with an increased risk of progression to cerebral malaria. The findings have implications for case and community management of childhood hyperparasitaemia and for malaria control efforts in sub-Saharan Africa where severe malaria is relatively common. Plasmodium falciparum hyperparasitaemia, defined as > 250,000 asexual parasites/μl blood or > 4% parasitized erythrocytes [hrocytes ,2, is a hrocytes ,4. Hyperhrocytes  but may hrocytes .P. falciparum to antimalarials including artemisinin derivatives and artemisinin-based combinations [P. falciparum malaria in a hyperendemic area of malaria in southwest Nigeria and describe the risk of hyperparasitaemia in these children.Delay in parasite clearance by all antimalarial drugs is associated with increasing parasitaemia and hyperparasitaemia ,7 and isinations . A receninations , an areainations , has shoP. falciparum is the predominant species accounting for 99% of all infections. Children are more affected than adults, and apparently asymptomatic infections occur in older school children and adults.The study was carried out between January 2008 and December 2010 in patients presenting at the Malaria Clinic at the University College Hospital in Ibadan, an endemic area for malaria in southwestern Nigeria [During the study period, all children with febrile illnesses suspected to be or clinically diagnosed as acute falciparum malaria were enrolled in the study after obtaining a written informed consent from parents or guardian of the children. Clinical evaluation consisted of a general clinical examination including measurement of weight, core temperature and physical examination. Core temperature was measured orally using an electronic thermometer. In very few young children it was measured rectally. Ethical clearance was provided by the local ethics committee.P. falciparum mono infections who were not included in 5 antimalarial efficacy trials (n = 2408) (see below) were treated with amodiaquine-artesunate and followed for approximately 2-4 weeks. Patients who met the inclusion criteria for enrolment into drug efficacy trials (n = 930) involving artesunate alone, mefoloquine alone, artesunate-mefloquine, artesunate-amodiaquine and artemether-lumefantrine were enrolled into these studies and have been reported elsewhere [Patients with parasitologically proven lsewhere -16.Thick and thin blood films prepared from a finger prick were Giemsa-stained and were examined by light microscopy under an oil-immersion objective, at × 1000 magnification, by two independent assessors (microscopists). A senior member of the study team reviewed the slides if there was any disagreement between the two microscopists. In addition, one in every four blood slides was reviewed by this senior member. Parasitaemia in thick films was estimated by counting asexual parasites relative to 1000 leukocytes, or 500 asexual forms, whichever occurred first. From this figure, the parasite density was calculated assuming a leukocyte count of 6000/mL of blood. Gametocytes were also counted in thick blood films against 1000 leukocytes assuming an average leukocyte count of 6000/mL of blood -19. HaemData were analysed using version 6 of the Epi-Info software , and theDuring the study period, 6807 children with illnesses suspected to be or diagnosed as malaria were evaluated. Parasitaemia was present in 3338 children, a parasite rate of 49%. The clinical characteristics of the 3338 children enrolled in the study are summarized in Table There was a significant positive correlation between body temperature and parasitaemia . There was also a significant relationship between log parasitaemia and body temperature . One of the 3338 children died from cerebral malaria; the child had no hyperparasitaemia at presentation.Eleven of 3338 children (0.3%) progressed to cerebral malaria: 7 of these had hyperparasitaemia and 4 had no hyperparasitaemia. The difference between the two proportions was significant . Seven of the children were < 5 year olds. The risk of progression was considerably higher in < 5 year olds with hyperparasitaemia , and DelIn contradistinction to the finding in an area of lesser intensity of transmission, for example, on the Thai-Burmese border, where hyperparasitaemia is associated with the dry season , there wAll the possible risk factors for hyperparasitaemia were not captured in the present study. Recent histories of diarrhoea and the presence of gametocytes, a finding associated with hyperparasitaemia in Thailand , for exaIn conclusion, a relatively broader age range, fever and enrolment after year 2008 are independent risk factors for hyperparasitaemia in malarious children in this endemic area. Hyperparasitaemia is significantly associated with an increased risk of progression to cerebral malaria in young children < 5 years old.The authors declare that they have no competing interests.AS led the design, conduct, data analysis and manuscript preparation, TMO was involved in conduct, data analysis and manuscript preparation, GOG and CTH were involved in design, conduct and preparation of the manuscript. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/268/prepub


Plasmodium vivax is the predominant species causes of malaria with about 90% total annual reported malaria in Iran. This study conducted to determine the susceptibility of Plasmodium vivax isolates to chloroquine in Sistan and Balochistan Province, southeastern Iran.P. vivax infection completed the designed 28-day in vivo study. The thick and thin film blood smears were screened for malaria parasites by microscopy. The nested PCR was applied using the Plasmodium 18 subunit ribosomal ribonucleic (Ssr RNA) genes for detecting mixed infections and diagnosis of parasites in the samples with low parasite on days 0, 5, 6, 7, and 28.A total 270 subjects with symptomatic malaria and confirmed P. vivax was cleared in 15%, 50%, 95%, and 100% of patients on days 1, 2, 3, 4 respectively by microscopy assessment. Six patients were exhibited specific P. vivax band in nested PCR on day 5. No recurrence was observed on days 7, 14 and 28. Mean (±standard deviation) parasite clearance time was 2.41 (±0.8) days.P. vivax is still susceptible to chloroquine in Southeatern Iran. This finding is compatible with results of neighboring countries Pakistan and Afghanistan. Plasmodium vivax (P. vivax) is the main cause of malaria in Asian, Central and South American countries  has been reported in Iran  stephancy, A. culcificis, A. fluviatalis, A. superpictus and A. pulcherrimus are the main malaria vectors in the area  infection.A total of randomly 285 subjects with symptomatic malaria and confirmed P. vivax mono infection with parasite density above 250/µl, and fever or history of fever during 48 hours prior to the time of recruitment. Patients with the following conditions were excluded from the study: presence of clinical condition requiring hospitalization, presence of severe malnutrition, pregnancy, chronic infectious diseases, and concomitant febrile illnesses. All patients were followed up for 28 days according to the WHO in vivo test protocol . According to the Iran national drug policy, primaquine (PQ) is given during 8 weeks for preventing relapses. Since half-life time of chloroquine extends nearly 30 days and during this time it can eliminate emergent parasites , thus PQ administration was postponed to day 28. Inclusion criteria for the registered malaria patients were patients who aged more than 1 year old, positive for protocol . The patP. vivax, which required hospitalization in presence of parasitemia, presence of parasitemia and auxiliary temperature of ≥ 37.5 at any time between days 3 and 28, and presence of parasitemia between days 7 and 28 irrespective of clinical conditions. Blood samples were taken on days 0, 5, 6, 7, 28 and stored at -20°C until DNA extracting for nested PCR. The study protocol was approved by the Tehran University of Medical Sciences, Iran. The consent obtained from all participants or their parents.The treatment failure was defined as clinical deterioration due to 3 of blood. Genomic DNA was extracted from the 200µL blood samples using a QIA quick PCR purification kit  according to the manufacturer’s protocol. The nested PCR was applied using the Plasmodium 18 sub-unit ribosomal ribonucleic (Ssr RNA) genes for detecting mix infections and diagnosis of parasites in the samples with low parasite (less 10 P/µl). Primers and detail PCR amplifying method have previously been described  were male. chloroquine was well tolerated and no patient reported vomiting within 1h of drug administration. Fever resolved on the first day in all subjects. According microscopy findings ectively . No recuPlasmodium genus on day 0. All 270 subjects showed ∼120 Bp band in the nested PCR, which is indicative of P. vivax malaria  and SD were 2.43  and 0.8 days respectively. MPCT based on sex was 2.39 and 2.44 days in males and females, respectively. There was no significant difference regarding MPCT between males and females . The number of parasite ranged from 1,000 to 35,000 P/ µl blood with geometric mean of 7,500 P/µl.The PCR product was consider positive when a band of size of ∼1200 Bp was observed for  malaria . Six  and Afghanistan , where Preported , while nreported , 20. OurP. vivax still has suitable susceptibility to chloroquine in southeast of Iran and chloroquine can be administrated for treatment of the vivax malaria patients at this region. Nested PCR was also suitable assay to determine exact malaria parasite clearance time in our in vivo study.In conclusion,


Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and their activation leads to the induction of effector genes involving inflammatory cytokines that may have contribute to controlling parasite growth and disease pathogenesis. The current immunogenetic study was designed to analyse the key components of innate immunity, TLRs and TIRAP (Toll-interleukin-1 receptor domain-containing adaptor protein), also known as MAL (MYD88 adaptor-like), in Iranian patients with mild malaria.tlr-4 (D299G and T399I), tlr-9 (T-1486C and T-1237C) and tirap (S180L) genes were assessed in 640 Baluchi individuals  from malaria-endemic regions using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods.The tlr-4 SNPs and promoter SNPs of tlr-9 were distributed among P. falciparum-infected and non-infected groups (P > 0.05) that showed no association of these variants with mild clinical manifestation. The comparison of the tirap S180L genotypes between patients with mild malaria and those healthy individuals showed that the frequency of heterozygosity was significantly higher in infected than non-infected individuals . The result also revealed a significant association of tirap S180L (P < 0.05) with development of mild malaria, which is common in Baluchi populations, who are living in malaria hypoendemic region of Iran but not in African populations (0%-6%).Common These data point towards the need for addressing the exact role of TLRs in contributing to human genetic factors in malaria susceptibility/resistance/severity within different malaria settings in the world. Plasmodium falciparum, ranging from asymptomatic infection to severe life-threatening forms. The development of severe malaria is due to the marked changes in cytokine expression that results in the individual's immune response to parasites. Indeed, the pathogenesis of severe malaria is complex and so far, the sequence of events leading to severe malaria is not completely known. To understand why some infections lead to severe forms of the disease, research has been focused on identifying parasite virulence phenotypes and the genetic make-up of the host [In malaria, clinical manifestations are different among individuals who are infected with the host .In vitro and in vivo results showed that TLRs  are central mediators of pro-inflammatory responses to Plasmodium infection [Toll-like receptors (TLRs) have been defined as pattern-recognition receptors whose function is to recognize pathogen-associated molecular patterns (PAMPs), including microbial pathogens, virulence factors and intracellular protozoan parasites -4. The anfection -12. The nfection .tlr-4, tlr-9 and tirap genes that alter susceptibility to infectious and inflammatory diseases [tlr-4, the most extensively studied genetic variations are two frequently non-synonymous co-segregating SNPs (D299G/T399I) that change the ligand-binding site of the receptor [tlr-4 polymorphisms are assumed to be correlated with risk of severe malaria as reported by Mockenhaupt and colleagues [tlr-9 promoter SNPs, T-1486C (rs1870884) and T-1237C (rs5743836) are assumed to be associated with susceptibility to asthma [tirap S180L SNP (rs8177374) has been reported to diminish TLR-2 and TLR-4 signaling [Single nucleotide polymorphisms (SNPs) have been described for diseases . For tlrreceptor . tlr-4 plleagues . Two como asthma  and placs D299G/T99I that ignaling  and heteL SNP rs87374 has TLR polymorphisms and malaria susceptibility due to differences in ethnicity, location, environmental factors and exposure to infectious pathogen; hence, conducting different studies on various populations are needed to clarify this issue. Therefore, the current study was designed to look at the polymorphisms in tlr-4 and tlr-9 genes in Baluchi ethnic groups from south-eastern parts of Iran with unstable and hypoendemic malaria transmission. In addition, SNP in tirap, which is a common adaptor protein for TLR-2 and TLR-4, was assessed. Finally, the frequencies of the common SNPs, observed in tlr-4, tlr-9 and tirap genes, among Baluchi patients with mild malaria and healthy control individuals were compared. The obtained results might enable us to understand the nature and outcome of the malaria disease in humans, and evaluate novel diagnostic and therapeutic strategies for this disease.Regarding the association between human gene polymorphism and disease, most studies for malaria susceptibility/resistance have been conducted on populations from Africa and south-east Asia. There is no information on malaria-associated gene polymorphisms from Iran, where malaria transmission is hypoendemic and there is no record of severe malaria and death due to this disease is rare. Up to this time, it is difficult to confirm the association between certain Plasmodium vivax as the predominant species and the second from October to November when both P. falciparum and P. vivax infections are usually recorded. In 2008, approximately 11,460 malaria cases were reported in Iran, 10% of which were microscopically diagnosed as P. falciparum. Most of the cases (94%) were recorded in the south-eastern provinces and in these regions; there is no report of severe malaria, anaemia or death due to malaria. Moreover, most patients were adults with uncomplicated malaria.Malaria is endemic in Sistan and Baluchistan Province (Iran), and the transmission is year-round with two peaks, the first from May to August with P. falciparum were recruited in an outpatient clinic at primary health centers in Chabahar district during April 2004-November 2009. These patients, who were presented with malaise, fever, or muscular pain and headache, were considered symptomatic and classified as having mild malaria  and parasitaemia was ranged 1,000 to 35,000 asexual parasites/mm3. All patients who were infected with P. falciparum had a fever and the diagnosis was confirmed via microscopy. Healthy Baluchi individuals  with no history of febrile or malaria clinical symptoms were included as control. Nested PCR was used to confirm infection and also co-infection with P. vivax in both patient and control groups. After obtaining an informed consent from adults or the parents or legal guardians of children, 1 ml of blood was collected from the individuals in vacuum EDTA tubes and stored at -20°C. All individuals with fever and confirmed malaria were treated according to the national guidelines. This study was approved by the Ethical Review Committee of Research in Pasteur Institute of Iran.The study population was consisted of 640 Baluchi individuals  from malaria endemic region of Sistan and Baluchistan Province. The patients (n = 320) with febrile Plasmodium species  was detected by nested-PCR amplification of the small sub-unit ribosomal ribonucleic acid (18ssrRNA) genes using the primers and cycling parameters described previously [Genomic DNA was isolated from whole blood collected in EDTA tubes, by using the commercially available DNA purification kit , and kept at -20°C until use. DNA of eic acid ssrRNA getlr-4 D299G and T399I, the two promoter SNPs of tlr-9 at positions T-1486C and T-1237C as well as tirap S180L SNPs were studied. The different SNPs were identified by PCR-RFLP analysis. The primers, PCR cycling conditions and RFLP are shown in Table 2, each of the four deoxynucleotide triphosphates at a concentration of 125 μM, and 0.2 U of Taq polymerase . The DNA fragments, obtained following PCR amplification or RFLP analysis, were electrophoresed on 2% to 3% agarose gels , respectively.The non-synonymous SNPs of tlr-4, tlr-9 and tirap genes using the primers described in Table To verify the results obtained by RFLP, all PCR products were sequenced for X2 test using SPSS for windows (version 16.0) and P < 0.05 was considered to be significant. To determine if genetic variation in tlr-4, tlr-9 and tirap genes were associated with risk of developing malaria, odds ratios (OR) and 95% confidence intervals (CIs) were calculated. The Hardy-Weinberg Equilibrium (HWE) was performed by comparing the observed numbers of different genotypes with those expected under the HWE for the estimated genotype frequencies [P > 0.05 was in HWE. Genotypic deviations from HWE were assessed using Pearson's chi-square (X2) statistical test.The sample size was calculated using OpenEpi software , with 80quencies  and P > P. falciparum, as a mono-infection, and none of the healthy control individuals had either P. falciparum or P. vivax infections.Based on nested-PCR results, a total of 320 patients with mild malaria were shown to be infected with P. falciparum-infected (n = 320) and non-infected (n = 320) Baluchi individuals were successfully analysed for the tlr-4, tlr-9 and tirap SNPs by PCR-RFLP methods and the sequencing data confirmed RFLP results. To determine whether there was any frequency difference in the allele (Table tlr-4 (D299G and T399I) and tlr-9 (T-1237C) were less than 10% and those for polymorphic alleles in tlr-9 (T-1486C) and tirap (S180L) were greater than 10% . In addition, two genotypes of tlr-4 T399T and tlr-4 T399I were observed among cases and controls with no significant difference (P > 0.05). tlr-4 I399I homozygous was absent in both studied groups  than among malaria patients  and non-infected cases (10.3%) with no statistically significant (P > 0.05). Furthermore, tlr-9 T-1237C frequency was low in the cases (1.6%) and controls (4.7%). The comparison of the tirap S180L genotypes between patients with mild malaria and those healthy individuals showed that the frequency of heterozygosity was significantly higher in infected than non-infected individuals   in Baluchi infected individuals than controls (33.8% vs. 25.6%). The comparison of healthy and infected individuals showed that the risk of mild malaria was increased 1.5-fold in individuals with tirap S180L . It is worth mentioning that the frequency of this heterozygosity was higher among Iranian infected patients with P. falciparum (33.8%), who are living in malaria hypoendemic areas (present study), than Ghanan, Gambian, Kenyan and Vietnamese populations (0%-3.5%) with reported severe cases of malaria [tirap S180L variant in Iranian malaria patients with mild malaria might support the role of this SNP in protection against severe malaria by intermediate levels of pathway activation and balanced inflammatory response .Study by Khor and colleagues  in Gambi-workers  in Brazi malaria . In facttirap L180L was found in both infected and non-infected Baluchi individuals (0.6% vs. 1%), which was not reported in African and Vietnamese populations, where malaria is highly endemic. The rarity of the mutant allele in African populations might be related with high mortality rate in these populations due to malaria, thus this may strongly reduce the frequency of tirap L180L genotype in these regions. This homozygousity suggested to be a risk factor for several diseases, including malaria [P = 0.653). The specific role of tirap L180L on malaria disease processes needs further investigation in different ethnic groups.Moreover, homozygous  malaria , which itlr-9 T-1237C. In addition, the observed genotype frequencies in the patients with mild malaria were significantly different from the expected frequencies for tlr-4 D299G, tlr-9 T-1237C and tirap S180L SNPs, which are deviated from HWE. This could be explained by genetic drift, non-random mating or an indication of selection acting on specific genotype and at the present, there is no any evidence to prove any of them in this population.Overall, wild-type homozygosity for all three examined genes was predominant among patients with mild malaria and healthy control groups. Statistical analysis also showed that all of SNPs were in HWE in the control group except for tirap S180L in malaria infected individuals point to the role of this genotype as a risk factor for the development of mild malaria, but protect from severe malaria. Comparing these data with previously reported results from other malaria endemic regions [TLRs and TIRAP in contributing to human genetic factors in malaria susceptibility/resistance/severity within different malaria transmission settings and manifestation pattern in the world.In summary, in the current immunogenetic study, it seems that the role of TLR polymorphisms is varying in different ethnic groups from different malaria endemicity. High frequency of heterozygous  regions ,25 suggeThe authors declare that they have no competing interests.SZ* designed the work, supervised the study, analysed the data and wrote the manuscript. SP carried out polymorphism studies. AAM contributed in the analysis of the data. NDD participated in sample collection, helped with the sequence data analysis and also critical reading of the manuscript. All authors read and approved final manuscript and agree to submission.


Plasmodium knowlesi is a potentially life-threatening zoonotic malaria parasite due to its relatively short erythrocytic cycle. Microscopic identification of P. knowlesi is difficult, with “compacted parasite cytoplasm” being one of the important identifying keys. This report is about a case of hyperparasitaemic human P. knowlesi infection (27% parasitaemia) with atypical amoeboid morphology. A peninsular Malaysian was admitted to the hospital with malaria. He suffered anaemia and acute kidney function impairment. Microscopic examination, assisted by nested PCR and sequencing confirmed as P. knowlesi infection. With anti-malarial treatment and several medical interventions, patient survived and recovered. One-month medical follow-up was performed after recovery and no recrudescence was noted. This case report highlights the extreme hyperparasitaemic setting, the atypical morphology of P. knowlesi in the patient’s erythrocytes, as well as the medical interventions involved in this successfully treated case. Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. Recently, P. knowlesi, a species naturally found in long-tailed macaque (Macaca fascicularis) and pig-tailed macaque (Macaca nemestrina) . This case report shows that A successfully treated hyperparasitemic knowlesi malaria case with atypical amoeboid morphology from peninsular Malaysia is described. In view of the challenges in diagnosis, healthcare workers should be aware of such atypical amoeboid morphology in knowlesi malaria samples. Besides, combination of artesunate and doxycyline can be an alternative treatment regime for knowlesi malaria cases. The renal function of knowlesi malaria patients should be monitored closely throughout the course of treatment, even after the parasites are successfully cleared from the blood stream.Consent was granted by patient for the publication of this case report.The authors declare that they have no competing interests.WCL, LCC, YLL, MYF and RM carried out laboratory works and analysed the data. PWC CJY and RRS collected blood samples, conducted clinical diagnoses and treatment intervention. WCL and YLL participated in the data analyses and helped to draft the manuscript. All authors read and approved the final manuscript.


Plasmodium ovale curtisi and Plasmodium ovale wallikeri were found together in time and space. ► Lack of recombination between them is not due to geographic or temporal separation. ► Therefore, the species barrier may be maintained by a biological mechanism(s).► We propose that two related species of malaria parasite cause ovale malaria. ► Discriminatory PCR tests identified both species in African surveys. ►  Plasmodium ovale curtisi and Plasmodium ovale wallikeri. It was recently shown that these two parasite types are sympatric at the country level. However, it remains possible that localised geographic, temporal or ecological barriers exist within endemic countries which prevent recombination between the genomes of the two species. Here, using conventional and real-time quantitative PCR (qPCR) methods specifically designed to discriminate P. o. curtisi and P. o. wallikeri, it is shown that both species are present among clinic attendees in Congo-Brazzaville, and occur simultaneously both in lake-side and inland districts in Uganda and on Bioko Island, Equatorial Guinea. Thus P. o. curtisi and P. o. wallikeri in these localities are exactly sympatric in both time and space. These findings are consistent with the existence of a biological barrier, rather than geographical or ecological factors, preventing recombination between P. o. curtisi and P. o. wallikeri. In cross-sectional surveys carried out in Uganda and Bioko, our results show that infections with P. ovale spp. are more common than previously thought, occurring at a frequency of 1–6% in population samples, with both proposed species contributing to ovale malaria in six sites. Malaria elimination programmes in Africa need to include strategies for control of P. o. curtisi and P. o. wallikeri.It has been proposed that ovale malaria in humans is caused by two closely related but distinct species of malaria parasite,  Since that time, relatively little attention has been paid to ovale malaria, which is considered to be uncommon, mild in clinical presentation and easily treated with the conventional antimalarial drug chloroquine , London, polymorphisms in six loci were examined in 55 isolates. Two distinct major haplotypes of each locus were identified and these did not recombine in any of the parasites examined. Thus P. ovale dimorphism was proposed to reflect the existence of two fully distinct ovale malaria species, which were unexpectedly shown to be broadly sympatric, at the country level, in both Africa and Asia  in Buliisa and Mayuge Districts, Uganda . Sampleson assay .2.1.3Plasmodium falciparum malaria with an estimated entomological inoculation rate >1000 infectious bites per person per year  and no other parasite species were identified by microscopy. DNA was extracted by the Chelex Method  assay of P. ovale parasites  in collaboration with the Ministry of Health and Social Welfare of Equatorial Guinea. As part of the evaluation of the BIMCP, annual malaria indicator surveys have been carried out since 2004 . During arasites .2.2potra  loci from field samples, the primers presented in Genomic DNA sequences were obtained as previously described  except tReal-time amplification assays were performed using the Quantitect Sybr Green PCR kit (Qiagen) on Corbett Rotorgene RG3000 and RG6000 thermo-cyclers, and output was measured by detecting SYBR green incorporation on the FAM fluorescence detection channel. PCR primers and cycling conditions were as previously described  or are gpo3gp locus, encoding glyceraldehyde-3-phosphatase, was carried out exactly as described  and the Ugandan National Council of Science and Technology granted ethics approval for the study in Buliisa and Mayuge Districts, Uganda. Informed consent was obtained in writing or by fingerprint (in cases of illiteracy) from all participating mothers on behalf of themselves and their children.Approval for the Bioko Island Malaria Indicator Survey was given by the ethics committees of the Equatorial Guinea Ministry of Health and Social Welfare, Malabo, and of LSHTM, UK . Written informed consent was given by each donor, or in the case of young children, by their parent or caregiver.Ethics approval for the facility-based survey in Apac, Uganda, was obtained from the medical biotechnology laboratories (MBL), the national ethical committee of Uganda and LSHTM, UK (project # 5539.09).Approval for the collection of blood samples in the Republic of Congo was obtained from the ethics committee at the Research Institute of Microbial Diseases, Osaka University , and sampling was authorised by the administrative authority of the Ministry for Research and the Ministry for Health in the Republic of the Congo. Written informed consent was obtained from individual patients and antimalarial treatment was provided for all individuals diagnosed with clinical malaria.33.1P. ovale on the basis of differential sizes of PCR amplification products, the recently described gene potra was amplified from a number of previously characterised P. o. curtisi and P. o. wallikeri isolates (P. o. curtisi (poctra) gene were identified , differing in six amino acids in the repeat region, as previously described. Two P. o. walliker (powtra) alleles were observed , differing in two non-synonymous positions. A small candidate amplicon encompassing the variable repeat region was delineated and amplified as described in P. ovale spp. were compared by agarose gel electrophoresis, and the two species were clearly distinguishable from each other , inland (Gamboma) and urban (Brazzaville) locations in the Republic of Congo. In total, 16 isolates had previously been identified as P. ovale sp. on the basis of microscopy, and seven additional samples were PCR-positive for P. ovale  in P. o. curtisi (pocrbp2) and P. o. wallikeri (powrbp2) (porbp2TMfwd and porb2TMrev (P. o. curtisi were replaced by cytosine (C) and guanidine (G) residues in P. o. wallikeri   were alirb2TMrev . In fiveallikeri A. We the species Aa, and i3.4Plasmodium spp. by LAMP , or as co-infections with P. falciparum (n = 2) or P. falciparum and P. malariae (n = 1). Two P. ovale spp. mono-species infections were incorrectly classified as P. falciparum infection by microscopy at densities of 320 and 360 parasites/μL. These six were comprised of five P. o. curtisi infections and one P. o. wallikeri infection.In Apac District, Plasmodium ovale spp.-positive samples from the surveys in Buliisa and Mayuge Districts were tested using the qPCR discrimination assay. Of 1850 individuals tested for malaria parasites of any species, 30 were identified as harbouring P. ovale: 11 with P. o. curtisi and 19 with P. o. wallikeri. In each case, species identity was confirmed either by the potra amplicon polymorphism assay or  by sequencing the pog3p P. ovale glyceraldehyde-3-phosphatase (pog3p) locus as previously described  and constitute strong evidence against the hypothesis that the two species are kept apart by mutually exclusive requirements for erythrocyte phenotype. Such continuing detailed surveillance will also provide the opportunity to further test our hypothesis that genetic recombination does not occur between the two forms of ovale malaria investigated here.An alternative explanation for the apparent lack of recombination between ovale malaria parasites, such as relapse periodicity (falciparum and vivax malaria (P. o. curtisi and P. o. wallikeri.It is now important to focus on determining whether there are any important biological, epidemiological or clinical differences between the two iodicity , drug re malaria , are als


Following the deployment of new recommendations for malaria control according to the World Health Organization, an estimation of the real burden of the disease is needed to better identify populations at risk and to adapt control strategies. The aim of the present study was to estimate the clinical burden of malaria among febrile children aged less than 11 years, before and after six-year of deployment of malaria control strategies in different areas of Gabon.Cross-sectional surveys were carried out in health care facilities at four locations: two urban areas (Libreville and Port-Gentil), one semi-urban area (Melen) and one rural area (Oyem), between 2005 and 2011. Febrile paediatric patients, aged less than 11 years old were screened for malaria using microscopy. Body temperature, history of fever, age, sex, and location were collected.Plasmodium falciparum-infection was the lowest in Port-gentil (below 10%) and the highest at Oyem (above 35%). Between 2005 and 2008, malaria prevalence dropped significantly from 31.2% to 18.3%, followed by an increase in 2011 in Libreville (24.1%), Port-Gentil (6.5%) and Oyem (44.2%) (p<0.01). Median age among the infected patients increased throughout the study period reaching 84 (60–108) months in Libreville in 2011 (p<0.01). From 2008, at all sites, children older than five years were more frequently infected; the risk of being infected significantly increased with time, ranging from 0.37 to 1.50 in 2005 and from 2.03 to 5.10 in 2011 in this group (p<0.01). The risk of being P. falciparum-infected in children aged less than five years old significantly decreased from 2008 to 2011 (p<0.01).A total of 16,831 febrile children were enrolled; 78.5%  were less than five years old. The rate of This study shows an increased risk of malaria infection in different areas of Gabon with over-five year-old children tending to become the most at-risk population, suggesting a changing epidemiology. Moreover, the heterogeneity of the malaria burden in the country highlights the importance of maintaining various malaria control strategies and redefining their implementation. Plasmodium falciparum risk distributions, and allows estimation of regional intensity of transmission, populations at risk, disease burden and level of prevention coverage [According to the level of malaria transmission and immunity acquisition, vulnerable populations differ in endemic areas. In highly endemic settings, children under five years and pregnant women are the most affected, constituting the main target population of new malaria control strategies as recommended by the World Health Organization (WHO) . In councoverage -11. Accocoverage . Howevercoverage .P. falciparum infection in children aged more than five years was reported, indicating the need to implement appropriate malaria control strategies. Previous surveys performed several years ago across the country showed differences in prevalence of malaria-positive blood smears (MPBS) ranging from 24% to 59% . With the support of the Global Fund, the Malaria National Control Programme (MNCP) organized the deployment of insecticide-treated bed nets (ITN) and artemisinin-based combination therapy (ACT) within the country. Indeed, in 2010, ITN coverage was 50% and ACT represented almost 99% of all antimalarial drugs prescribed in public health structures in Gabon [In Libreville, the main urban city of Gabon, a reduction of malaria prevalence has been observed following implementation of new control strategies . A trendProspective cross-sectional surveys were conducted between 2005 and 2011 in Gabon, at Libreville the capital city and Melen one of its suburban area, at Port-Gentil the second largest town of the country and at Oyem. Recruitment was carried out at four health care centres: i) the Malaria Clinical and Operational Research Unit (MCORU) in Libreville, the capital city of Gabon; ii), the regional hospital of Melen (HREM), a semi-urban area located at 11 km north of Libreville; iii) the regional hospital of Oyem (CHRO), a rural northern area; and, iv) the regional hospital of Port-Gentil (CHRP), an urban area located on a peninsula on the west coast. The first three sites were selected as sentinel sites for malaria survey by the Malaria National Control Programme (MNCP). The CHRP was selected because previous data highlighted a low frequency of confirmed malaria cases in private health structures that were more frequented by patients than public health services in the city. The CHRP was therefore built by the Ministry of Health to facilitate access to health care for the low-income population. Oyem is a town surrounded by the forest with a level of urbanization being the lowest compared to Libreville, the capital city, Port-Gentil, the economic city, and Melen which benefits from its proximity to Libreville; people are living in villages located at less than one Km from the CHRO. Moreover, the main economic activities are related to agriculture and performed by the main population living at Oyem and in the surrounding areas. Indeed, workers in public administration are less represented.P. falciparum is perennial without significant fluctuation throughout the year in Gabon, the study team of MCORU collected data during at least one rainy and one dry season in each site. In 2011, during the study period, the recruitment at Oyem included out and inpatients, as well as febrile patients who were prescribed a biological analysis in private clinics since laboratory of the hospital was the only structure that could realize all the biological examinations at this time.As malaria transmission, predominantly caused by P. falciparum infection, therefore data collection was part of routine activities at the CHL, at Melen, and at Oyem where MCORU team was committed to set up the sentinel site activities according to WHO recommendation. At Port-Gentil, members of the study team took part in emergency and paediatric ward consultations and, during the study period, all the attending febrile children were screened for malaria.In sentinel sites, febrile patients are routinely screened for Febrile paediatric outpatients and inpatients, aged less than 11 years old were included. Body temperature, history of fever, age, sex, bed net use, home treatment with anti malarial and location were collected. Data on self-medication have been collected through a detailed CRF. Patients were asked about previous history of fever and drug intake the month before the consultation, the type of molecule taken and the duration of the treatment during each survey throughout the study period.For each patient, a thick blood film was done according to the Lambaréné procedure and read as described previously . ParasitA febrile case was defined for any child with axillary temperature ≥37.5°C or history of fever during the 48 hours prior to the day of consultation. Malaria case was defined by fever associated with a malaria positive blood smear (MPBS).P<0.05 were considered significant. All reported p-values are two-tailed. According to the sample size calculation, the number of patients recruited was always higher than the minimum required, although the total number of patients screened at Libreville and Port-Gentil in 2011 was lower compared to that of the other sites. Sample size was calculated taking into account previous prevalence of P.falciparum microscopic infection obtained in 2005 or 2008 according to the study areas. It was based on 5% precision with 95% level of confidence, using finite population correction according to NAIN (2006). Minimal sample size was 240 for Oyem, 199 for Libreville, 308 for Melen and 88 for Port-Gentil.All data were entered and cleaned using Epi-info version 3.3.2 (2005 CDC Atlanta). Analysis was performed using Statview 5.0 . Proportion of malaria patients were compared using Pearson Chi-square and means or median by either Mann-Withney or Krushkal-Wallis non-parametric tests, according to site and time point as appropriate. Multiple comparisons of malaria frequencies according to site, age and year were performed by using a log rank test (Mantel Haenzel). A logistic regression analysis stratified by age groups (<5 years/>=5 years) was performed to assess the effect of site on malaria infection. The effect estimates are the odds ratio with a 95% confidence interval. Analyses resulting in values of Malaria diagnosis, anaemia detection, drug resistance monitoring and interventions coverage in sentinel sites are the main strategies for malaria control of the Gabonese Ministry of Health (MH) represented by the Malaria National Control Program (MNCP). The Department of Parasitology-Mycology (DPM), is the reference laboratory for malaria diagnosis and anti-malarial drug resistance evaluation. The DPM is committed by the MH to carry out these evaluations throughout the country in order to provide reliable data for policy adjustment in collaboration with MNCP. All data obtained are part of routine activities in sentinel sites and at CHRP that are under the administrative supervision of the MH. Oral consent was obtained from parents or legal guardians for data publication. Each patient with a MPBS was treated according to national recommendations, using ACT, free of charge for children with uncomplicated malaria.A total of 16,831 febrile children were enrolled: 78.5%  were less than five years old. Throughout the study period, the median age was the lowest in 2005, below 24.0 (12–48) months. In Libreville and Melen, the urban and semi-urban areas, the median age of the febrile patients did not vary between 2005 and 2011, although the IQR was larger in 2011  varied along the time. Between 2005 and 2008, a significant drop in malaria prevalence from 31.2%  to 18.3% , then an increase to 37.9%  in 2011 was observed (p<0.01). Statistical analysis showed a significant increase of malaria prevalence from 2008 to 2011 in Libreville, Port-Gentil and Oyem, whereas it slightly decreased from 2005 to 2011 at Melen (p<0.01)  (Table p<0.01). In 2011, it was the highest among children older than five years old at all sites compared to the previous years. Logistic regression showed a significant risk of being infected according to site and age (Table The median age among the infected patients increased throughout the study period p<0.0 (Table 11) Table . The risge Table .p <0.01)  in 2008 to 1.45 (1.21-1.73) in 2011 (p<0.01).Among children aged less than five years, a significant decrease of malaria cases was observed in most of the sites, except at Port-Gentil, during the study period (1) Table . Indeed,Overall, the median parasite density (MPD) was 8,500  P/μL. During the screening period, the MPD increased from 9,000  P/μL to 14,700  P/μL at Libreville and from 84 (70–168) P/μL to 5,600  P/μL at Port-Gentil. Infected children from Oyem and Libreville carried the highest MPD: 15,085  P/μL at Oyem and 6,300  P/μL at Melen.P. falciparum infection prevalence between 2005 and 2011. Large differences were observed between the sites, highlighting the heterogeneity of the epidemiological features of malaria in Gabon, even for sites that are geographically close together . Indeed, between, Melen and Libreville, the difference in the proportions of infected patients is lower probably due to the fact that health care facilities in both sites are accessible to all patients. Moreover, the difference between the patients consulting at both structures when considering the socioeconomic level is not so obvious, although, Melen is a sub-urban area of Libreville characterized by a majority of slums with a low income population, compared to the population of Libreville that is more complex in regards to the different living conditions.In areas with high level of malaria transmission, children under five years and pregnant women are the most vulnerable population and the main target of prevention strategies. The present study, carried out at four health care facilities in several areas of Gabon, estimated the evolution et al.[As already described in urban cities, the prevalence was lower in coastal urban areas of Libreville and Port-Gentil. It was much higher at Libreville, probably due to a difference in urbanization level, equipment, housing and access to treatment between both cities. These differences do not seem to be related to ACT self medication and bed net coverage as reported elsewhere . In all et al. could beP. falciparum infection rates already observed in Libreville, is also confirmed in other areas of the country [Previous studies reported a decline in malaria prevalence in febrile children over time ,14,18,19Plasmodium carry a high proportion of molecular markers of resistance to the commonly used insecticides, with an impact on the effectiveness of the current vector control programs [Anopheles gambiae s.s. and Anopheles funestus s.s. were controlled effectively with the ITN and IRS programme, maintaining a reduced disease transmission and burden [After the decline in the proportion of malaria cases at all sites between 2005 and 2008, a rebound in infection rates in rural and urban areas appears. In the same time, a slowdown in the prevention activities led by the MNCP and a low ITN coverage may partly explain this phenomenon. As an example, in Malawi, from 2001 to 2005, the proportion of malaria cases decreases of 50% 34.5% to 17.1%), followed by a rebound to 25%. An association with the slowing of interventions from 2001 to 2010 is characterized by a reduction of free ITN distribution, a limited free access to ACT in health care facilities for vulnerable populations, the lack of re-impregnation of used bed nets and indoor residual spraying (IRS) . In Keny.1%, folld burden . EnvironAnopheles population in both cities [Consistent with other tropical areas, children under five years constitute the majority of patients consulting for fever . The proh cities ,28. In Th cities . In Tanzh cities . The youh cities . Similarh cities .Together, the rates of malaria prevalence and the shift in the age of at risk children evoke a changing epidemiology of malaria in Gabonese cities, which cannot be all considered as malaria hyperendemic areas. Although the transmission is perennial, it rather becomes meso-endemic in some towns such as Libreville and it is much lower at Port-Gentil. Cohort studies and new entomological surveys from other areas are urgently needed to further characterize the malaria transmission in the country.This study has some limitations. Patients could not be screened at all the sentinel sites. Otherwise, this study is a health centre survey. Therefore, it does not represent the situation in the whole population but it already provides reliable important data. Furthermore, assessment of bed net effective use and type , and their direct impact on malaria prevalence cannot be established.Baseline epidemiological data are necessary to develop appropriate malaria control strategies and public health policy. This study shows that despite a decline of malaria cases observed in several endemic areas of Gabon, there is an increased risk of malaria infection among children older than five years who are becoming the most at-risk population. The heterogeneity of malaria epidemiology in Gabon underlines the need to increase, to maintain the various malaria control strategies and to redefine their implementation on a continual basis, particularly in urban areas where a rebound in malaria is being observed. Further entomological surveys in rural and semi-urban areas are needed in order to better support malaria prevention and control. This work has important implications for the planning of future campaigns based on the distribution of long-lasting, insecticide-treated nets and the introduction of intermittent preventive treatment for children in GabonThe authors declare that they have no competing interests.The study was initiated by MK. DPMM and MKBA conducted the study with the contribution of all authors. DPMM and MKBA interpreted the data and drafted the manuscript. Data were analyzed by MKBA and EK. NJ, MOM, MJR and MCRU team participated to the field study. All authors revised and approved the final manuscript.


Plasmodium falciparum malaria parasites in Pailin province, along the border between Thailand and Cambodia, have become resistant to artemisinin derivatives. To better define the epidemiology of P. falciparum populations and to assess the risk of the possible spread of these parasites outside Pailin, a new epidemiological tool named “Focused Screening and Treatment” (FSAT), based on active molecular detection of asymptomatic parasite carriers was introduced in 2010. Cross-sectional malariometric surveys using PCR were carried out in 20 out of 109 villages in Pailin province. Individuals detected as P. falciparum carriers were treated with atovaquone-proguanil combination plus a single dose of primaquine if the patient was non-G6PD deficient. Interviews were conducted to elicit history of cross-border travel that might contribute to the spread of artemisinin-resistant parasites. After directly observed treatment, patients were followed up and re-examined on day 7 and day 28. Among 6931 individuals screened, prevalence of P. falciparum carriers was less than 1%, of whom 96% were asymptomatic. Only 1.6% of the individuals had a travel history or plans to go outside Cambodia, with none of those tested being positive for P. falciparum. Retrospective analysis, using 2010 routine surveillance data, showed significant differences in the prevalence of asymptomatic carriers discovered by FSAT between villages classified as “high risk” and “low risk” based on malaria incidence data. All positive individuals treated and followed-up until day 28 were cured. No mutant-type allele related to atovaquone resistance was found. FSAT is a potentially useful tool to detect, treat and track clusters of asymptomatic carriers of P. falciparum along with providing valuable epidemiological information regarding cross-border movements of potential malaria parasite carriers and parasite gene flow.Recent studies have shown that  Plasmodium falciparum to antimalarial drugs since the 1960s P. falciparum AR parasites was reviewed P. falciparum AR parasites, the “Artemisinin-Resistance Containment Project” was implemented by the governments of Cambodia and Thailand with coordination and technical support by WHO and partners with funding from the Bill & Melinda Gates Foundation. Its overall goal was to contain artemisinin-tolerant P. falciparum parasites by removing selection pressure and reducing and ultimately eliminating falciparum malaria. In order to achieve this goal, an ambitious programme of activities was carried out, including the provision of universal coverage with long-lasting insecticide treated nets (LLINs) and of rapid access to free, parasitologically confirmed diagnosis through rapid diagnostic test (RDT) and ACTs delivered by community-based Village Malaria Workers (VMWs).The border region between Cambodia and Thailand, specifically in Pailin province, has repeatedly been the epicenter of emerging resistance of P. falciparum asymptomatic carriers. PCR-based cross sectional screenings were focused on villages with the highest annual incidence rates for acute P. falciparum malaria infections reported by heath centers (HC) through malaria microscopy and by VMWs using RDT. Based on high-throughput molecular diagnosis of malaria, the present paper compiles information obtained through the FSAT pilot with a view to gaining more micro-epidemiological knowledge about the population of asymptomatic carriers which would otherwise have remained largely undetected.Initially, a much wider operation referred to as “Mass Screening and Treatment” (MSAT), which aims at screening and treating the entire population of Pailin, estimated at over 70,000, within two weeks was envisaged but abandoned following two pilot studies conducted in November 2008 and May 2009. It was therefore decided to carry out to evaluate Focused Screening and Treatment (FSAT), a PCR-based strategy intended to detect P. falciparum in particular was highly clustered in Pailin, the 109 villages of Pailin were split into two groups: “high risk” villages and “low risk” villages based on available records of parasitologically confirmed symptomatic cases of P. falciparum malaria (2009). The “high risk” village group consisted of the 10 villages reporting the highest P. falciparum incidence rates at HC and of 8 villages reporting the highest number of cases through VMWs in 2009. All others were classified as “low risk” villages. As three villages of Pailin qualified for “high risk” in both categories (HC and VMW records), only 15 out of Pailin's 109 villages were identified as “high risk”, the remaining 94 were classified as “low risk”. All 15 “high risk” villages were selected, comprising among them 71% of all parasitologically confirmed cases of P. falciparum malaria recorded in Pailin in 2009 (623/941). The 2009 village selection threshold between “high risk” and “low risk” villages was >8 cases of P. falciparum with high risk villages reporting 9 to 78 (average 41.5) cases and low risk reporting 0 to 7 (average 3.4) cases throughout 2009 . The “high risk” villages included seven villages  classified as “high risk HC 2009”, five villages  classified as “high risk VMW 2009” and three villages  classified as “high risk HC/VMW 2009”. Five villages  were randomly selected among the remaining 94 of 109 villages, classified as “low risk 2009” . Procedures to screen individuals were divided among 4 dedicated stations:FSAT interventions were conducted at a rate of 3 villages per month. The Screening Team (ST) of seven persons spent four days in each village. The ST was followed at a one-week interval by the Follow-up and Treatment Team of two persons (FTT). Both ST and FTT received local support by the active participation of the village chief in identifying the individuals participating as well as the VMW assisting the direct observation of treatment of those found positive by RDT or PCR. The ST proceeded to the next village and so on until it had screened three villages, before taking one week's rest. During the 4Two ST members, assisted by the village chief, located each villager in the existing village register. The villager's name was manually transcribed into the FSAT screening register with a unique ID code (village number+family number+letter designating position in the family+screening sequence number). Absent family members were also recorded. In order to assess the risk of spread of drug resistance, the individuals were questioned regarding travel over the past year and classified as “risk class A” (travel to or from Myanmar), “risk class B” (travel to or from Thailand), “risk class C” (travel within Pailin Province only) and “risk class D” (travel elsewhere within Cambodia). The screening process was explained to each villager, prior to signing or thumb-printing of the consent form.Axillary temperature was recorded for each participant and the result transmitted immediately back to the registration and informed consent station. Individuals with temperature ≥37.2°C received an additional mark on their arm, indicating that they would require a malaria rapid diagnostic test (RDT).Any individuals detected as being sub-febrile or febrile were tested for malaria by using a RDT , detecting falciparum malaria (HRP-2) and non-falciparum malaria (lactate dehydrogenase). If the result was positive, a mark was made on the villager's forearm, identifying him/her for the malaria treatment station. For all participants in the survey, finger prick blood samples were collected and used to perform thick and thin films and double blood spots . For quality control, in each 96 well-format PCR plate, one field positive and one negative blood spot controls was included by the ST and blinded to laboratory staff at the Pasteur Institute in Cambodia (IPC) in Phnom Penh, which carried out the PCR tests.P. falciparum with atovaquone-proguanil  according to manufacturer's instruction or with CQ for non-P. falciparum infections , according to the national treatment guideline. Prior to treatment, pregnant women were detected through questioning and a pregnancy rapid diagnostic test, if necessary. Pregnant women received quinine . As soon as the patient received the first dose of treatment, a detailed follow-up interview was conducted. RDT negative cases received multivitamins and anti-helminthic treatment, and if symptomatic with fever and other clinical signs of bacterial infection, co-trimoxazole. At the completion of screening in each village, slides and dried blood spot samples were transported within 24 hours to the IPC laboratory in Phnom Penh.At the treatment station, RDT positive cases were treated for b gene and G6PD status was transmitted to the FTT from IPC by SMS. PCR-confirmed cases of P. falciparum infections were treated with AP in absence of mutation at codon 268 in cytochrome b gene. Direct observed treatment at dosages following manufacturer recommendation was carried out over three days. Malaria cases were followed up daily for the first three days and on day 7 and day 28. Primaquine (PQ) was used according to G6PD status. Individuals with normal G6PD levels and infected with P. falciparum received a single dose of PQ  under supervision on day 2, while normal G6PD infected by P. vivax received CQ over three days according to WHO guidelines and PQ for 14 days (0. 50 mg/kg). Any patients presenting with severe symptoms were given emergency support and referred to the Pailin referral hospital for further assessment and management. Blood was obtained by finger prick on all follow-up days and on any unscheduled day to use for analysis of thick and thin blood smears and for storage on filter paper.Follow-up was triggered when the list of PCR positive cases including species identification, identification of Y268N mutation in cytochrome P. falciparum recrudescent cases. DHA-PIP  was used for non-P. falciparum recrudescent cases.Treatment outcomes were classified as treatment failure if the patient presented with recurrent asexual parasitemia at day 7 or day 28 and treatment success if both day 7 and day 28 asexual parasitemia were negative. According to the national treatment policy, quinine  plus tetracycline  was used at Pailin referral hospital to treat Microscopy blood slides were stored in order to cross-check all RDT and PCR positive samples. Blood smears were stained with 3% Giemsa for 30 min at room temperature. Asexual- and sexual-stage parasite counts were performed by two experienced microscopists. In the case of a discordant result, a third reading was done. Parasite densities were determined from thick blood smears by counting the number of asexual parasites per 200 WBCs , assuming a WBC count of 8,000/µl. A smear was considered negative if no parasites were seen after review of 100 high-power fields.al.DNA from blood spot collected in 96-well PCR plate format containing patient screening samples and field positive and negative controls were extracted with Instagene Matrix resin® , according to the protocol adapted from the suppliers recommendations and previously described by Steenkeste et Plasmodium was carried out using primers targeting the Plasmodium cytochrome b gene as described by Steenkeste et al.Plasmodium species. Briefly, 5 µl of PCR products adjusted to 500 ng/µl were mixed with 0.2 µl of AluI and 2.5 µl of buffer A according to the manufacturer's instructions , incubated for 4 hours at 37°C and inactivated at 65°C for 15 minutes. Bands were detected by standard 2% agarose gel electrophoresis and ethidium bromide staining. Plasmodium species identification was assessed according to the number and the size of the bands: P. falciparum , P. vivax , P. ovale  and P. malariae .Detection of P. falciparum positive samples were screened for the Y268 mutations in cytochrome b gene that have been related to atovaquone resistance. PCR products from the outer PCR used to detect malaria parasites (1385 pb) were used as template for two second round PCRs. For both nested PCR (NsiI and SspI), amplification took place in the following reaction mixture: 2.5 µl of 10× buffer, 1.5 mM MgCl2, 0.2 mM each deoxynucleoside triphosphate, 0.5 µM each primer , 2 U of FirePol Taq polymerase , and 2 µl of PCR products. Nested PCRs were performed under the following conditions: heating at 94°C for 5 min, followed by 30 cycles of heating at 94°C for 30 s, 45°C (nested PCR NsiI) or 55°C (nested PCR SspI) for 90 s, and 72°C for 2 min, and a final extension period at 72°C for 10 min. Five µl of nested PCR products adjusted to 500 ng/µl were respectively mixed with 0.2 µl of NsiI and 2.5 µl of buffer 3 (nested PCR NsiI) and with 0.2 µl of SspI and 2.5 µl of buffer 2 (nested PCR SspI) according to the manufacturer's instructions , incubated for 4 hours at 37°C and inactivated at 65°C (SspI) or 80°C (NsiI) for 20 minutes. Bands were detected by standard 2% agarose gel electrophoresis and ethidium bromide staining. Polymorphism at codon 268 was assessed according to the number and the size of the bands: Y268Y (tat) - NsiI RFLP  and SspI ; Y268N (aat) - NsiI RFLP  and SspI ; Y268C (tgt) or Y268S (tct) - NsiI RFLP  and SspI .All Plasmodium species identification and mutations at codon 268 were secondly confirmed by sequencing. Briefly, nested PCR products were purified by 96-well plate filtration  using a polyacrylamide gel . Sequencing reactions were carried out with the ABI Prism BigDye Terminator cycle sequencing ready reaction kit run on a 3730 XL genetic analyzer . Electrophoregrams were visualized and analyzed with Seqscape software v2. 0 . Amino acid sequences were compared with the following sequences: GenBank accession no. NC002375 for P. falciparum, AY598138 for P. vivax, NC007232 for P. knowlesi, AF069624 for P. malariae and AB182496 for P. ovale, using BioEdit Sequence Alignment Editor software alTo determine which individuals should receive primaquine treatment, all individuals who tested PCR positive were screened for G6PD deficiency from backup blood spots according to the protocol developed by Kuwahata et msp-1), merozoite surface protein 2 (msp-2) and glutamate rich-protein (glurp) using nested-PCR as described elsewhere msp1, msp2 and glurp alleles present at the time of failure had been present at the time of treatment initiation. In all other cases, the failure was considered a new infection.Molecular genotyping techniques were used to distinguish recrudescence from new infection for all patients who failed therapy after day 7. Filter paper blood samples collected on the day of enrolment and on the day of failure were analyzed for polymorphisms in the genes for merozoite surface protein 1  by nested PCR approach  of less than 0.05 was considered statistically significant.Data were entered and verified using Microsoft Excel software® and analyzed using XLSTAT for Windows XP®  and MedCalc software . The Mann-Whitney U test or Kruskal-Wallis method were used for non-parametric comparisons, and Student's t test or one-way analysis of variance for parametric comparisons. For categorical variables, Chi-squared or Fisher's exact tests were used to assess significant differences in proportions. The efficacy outcomes were 28-day clinical and parasitological failure risks adjusted by genotyping, in accordance with the 2009 WHO protocol He) from haploid data and calculated as He = [n/(n−1)][1−pi]  and by the Ethics Board of the WHO Western Pacific Region office (WPRO). An informed written consent was provided by the parents/guardians of all patients before testing.P. falciparum or mixed P. falciparum/non-P. falciparum species (40 and 120 parasites/µL) and 5 with non-P. falciparum species . Ten additional malaria cases were found by using PCR : 3 with P. falciparum  and 7 with P. vivax . Detailed information including microscopic results is presented in The FSAT study was conducted from May to October 2010. A total of 6,931 individuals were screened in the 20 selected villages. The coverage achieved was 72.7% when counting all persons present at the village at the moment of FSAT and their relatives who were known but absent . The mean age of the screened population was 23.7 years , ranging from 1 week to 89 years. The mean gender ratio  was 1.04, ranging from 1.32 (in Oh Tatus and Oh Trengleu villages) to 0.88 (Bor Thmey). Risk classification according to the movement of individuals showed that none of individuals declared that they had come from or were be likely to go to Myanmar (“risk class A”); 139 (1.6%) were identified to have a travel history to or from Thailand and were classified as belonging to “risk class B”; 8550 (94. 6%) declared that they had stayed or were likely to stay within Pailin or local transmission areas within Cambodia (“risk class C”); and 341 (3.8%) declared that they had come from or were likely to proceed to non-malaria transmission areas within Cambodia (“risk class D”). Three individuals (1 in Phnom Dambang and 2 in Oh Preus) were said to work as maids in Malaysia and were initially falsely classified as “risk class A”. However, given that they were most likely to work in an urban area they were considered not be posing a risk to the spread of malaria parasites from Pailin, they were removed from “risk class A” and remain unclassified. No significant differences were observed between villages with regard to risk classes. In the 20 villages, 621 individuals (8.9%) were febrile or sub-febrile with axillary temperature ranging from 37.2°C to 40.0°C, with a mean temperature of 37.4°C. Among them, only 7 (1.1%) were detected positive by RDT: two with P. falciparum , 72 by P. vivax  and one by P. malariae . The mean time between sample collection and treatment was 8 days (SD±4 days).Analysis of 6931 dry blood samples by nested-PCR showed 133 (1.9%) positive samples : 60 indiP. falciparum (P = 0.0001) and P. vivax (P = 0.0003)  . All malP. falciparum infections were found in 9 villages . The prevalence of P. falciparum infections was not significantly different between adults (0.94%) and children under the age of 15 (0.74%) (P = 0.36) and between males (1.02%) and females (0.72%) (P = 0.18).Sixty vs. 0.59%, P = 0.41). In contrast, there was a statistically significant correlation between the mean prevalence of parasite carriers of “high risk” and “low risk” villages identified by VMW in 2009 . While the FSAT investigation was being conducted from May to end of October 2010 detecting almost exclusively asymptomatic parasite carriers, the disease surveillance system in Pailin continued to detect and parasitologically confirm the incidence of symptomatic carriers. At the end of the year a re-classification of high and low risk villages became possible based on the newly available incidence data of 2010 using the same selection criteria as for 2009 incidence reports. Even though the reported incidence of P. falciparum had fallen by 73.4% (from 941 to 250) in 2010, 11 out of the initial 15 high risk villages remained among the ten with highest case reports in the two HC and VMW disease surveillance systems . The analysis of the FSAT results showed that the correlation between the prevalence of asymptomatic carriers and disease incidence reports (both surveillance systems) of the same year was highly significant. “High risk HC 2010” villages had on average six times more asymptomatic carriers than “low risk HC 2010” villages . A very similar result was observed when comparing “high risk VMW 2010” villages with “low risk VMW 2010” villages  . P. falciparum prevalence was not significantly different between adults and children  and between males and females . The spatial distribution of P. falciparum prevalence according to malaria incidence reported through HC and VMW in 2009 (Panel 1) and in 2010 (Panel 2) is shown in According to the classification of villages, the mean prevalence of asymptomatic parasite carriers was not significantly different between “high risk” villages and “low risk” villages based on HC 2009 data (0.38%  P<10−4) . Based oP. falciparum isolates displayed 17 different haplotypes. Twelve haplotypes (70%) were found solely in several villages: Pekiri , Bor Thmey , Andong Thmor (N°8 & 16) and Oh Chrakandal (N°15) while the others were shared between villages: N°1 (Oh Sour Sdey & Pekiri), N°5 (Bor Tang Sou & Pekiri), N°6 , N°7 , N°10 . Genetic diversity, assessed by Nei's unbiased expected heterozygosity (He) ranged from 0.06 (Oh Roel) to 0.33 (Bor Thmey) and was significantly higher in Bor Thmey compared to Pekiri , highlighting a parasite flow between these two villages.Eight-locus genotyping of P. vivax infections were found in 12 villages (8 out of the 20 villages screened had no P. vivax cases). P. vivax infections were significantly more prevalent in adults , but no significant difference was found between males and females . Spatial distribution of P. vivax prevalence is shown in Seventy two Screening of G6PD deficiency by using blood spots among the malaria infected cases gave results which were considered skewed towards artificially low G6PD enzyme activity levels due to methodical problems, as explained in the discussion section. The global prevalence of G6PD deficient people among parasites carriers was estimated to 63% (18% with 10%–30% of enzyme activity and 45% <10% of enzyme activity).P. falciparum cases, all parasites were wild-type (Y268).Among the 60 P. falciparum cases: AP (n = 25), AP+PQ (n = 25), CQ , QN  and DHA-PIP . Three individuals were lost . The 28-day follow-up data were available for 51 patients. One patient treated with DHA-PIP failed and was classified as treatment failure following genotyping. One patient treated with AP developed vivax malaria at day 28.Five different antimalarial regimens were given to 57 of 60 P. vivax cases, 59 patients received CQ alone and only 11 patients received CQ with PQ due to the many false positive G6PD tests. Two individuals were lost . The 28-day follow-up data were available for 64 patients. Both CQ and CQ+PQ were 100% effective in clearing P. vivax infections.For The emergence of artemisinin resistance in western Cambodia demanded urgent action to contain its spread and eliminate malaria in identified foci The most encouraging epidemiological insight gained from FSAT was to that seems extremely low. Out of the 6,931 persons screened and among the 2,606 family relatives of the screened who were absent at the time of screening, none had any travel history to or from Myanmar. Although there is long history of presence of Burmese gem miners in Pailin P. falciparum spread outside of Pailin. However, temporary migrations of seasonal workers from Cambodia to Thailand seem to be likely responsible for the parasite gene flow that we have observed between villages along the Cambodian-Thailand border (i.e. Bor Thmey) and villages inside Pailin province (i.e. Pekiri).There had also been concerns that artemisinin resistance might spread indirectly from Palin to Myanmar via temporary or permanent residents of Pailin when migrating to areas of local transmission on the Thailand side of the border, to work in orchards, rubber or plantations or in the fishing industry at the coast in which many Burmese are working Until the launch of the FSAT project, there were concerns that the existing health information system (HIS), and in particularly the HC incidence data would not accurately identify foci of infection because it is known that patients in Cambodia seek treatment in the private sector, especially if they are far away from public health facilities The VMWs are clearly a critical component of the containment strategy. As shown here, not only are they instrumental in reducing population parasite burden by providing easy access to good quality diagnosis and treatment, but they are a critical component of the surveillance system P. falciparum cases that were detected by PCR, 95% could be traced for follow-up. Of those who could remain in the study all adhered to the directly observed three days of treatment. Of these cases, 96% were followed up until day 28. P. falciparum parasites circulating in Pailin province were found to be sensitive to atovaquone (none of them being mutant-type at codon 268) and asexual parasites totally sensitive to AP. Only one case of treatment failure was observed from an individual treated with DHA-PIP. This observation was not surprising due to high level of recrudescence (∼25%) recently found in this area .FSAT proved achievable logistically, the complex protocol of screening, treatment and follow up of over 6'000 individuals was sustained over six months. In addition, FSAT has generated scientific results on patterns of infection and quantified the risks of potential drug resistant parasites spread outside of Pailin. This operational success under difficult field conditions suggests that FSAT might be a feasible method for cross-sectional screening with PCR and directly observed treatment in other settings. A high level of case identification and treatment was achieved despite there being a mean lapse of 8 days between the day of screening and the case being located by the follow-up team. Out of the b gene and G6PD deficiency screening can now be similarly carried out within the same time and expected increases in reliability. Moreover, the blood sample analysis within the village is likely to render this practice more acceptable and interesting for the population, increasing the coverage rates.A number of important lessons were learnt which may help to inform the future programme implementation. The main challenge was the lack of accurate information on the size and location of the population because of the amount of population movement in the area. As noted elsewhere, much of this movement is dictated by the seasonal agricultural calendar P. falciparum from the province. The arguments in favour of MDA include the slowness of FSAT (screening speed of one village per week) and the possibility that there would be asymptomatic carriers under the low parasitaemia threshold of PCR of 0.1 parasites/µl who might escape detection. However, the main difficulty encountered during FSAT  would also be encountered by MDA. But, in contrast to FSAT and MSAT, MDA would also make any epidemiological analysis impossible and effectively render the programme blind as to the location of last remaining foci of transmission. Finally, the rapid geographical widening of drug pressure caused by MDA, followed by a longer period of mass exposure of sub-lethal plasma concentrations of the drug used, present simultaneously in large sections of the population, could trigger the very selection and emergence of multi-drug resistant parasite populations that many so rightly fear. This would be particularly true for AP if it was to be used for MDA, as only one single mutation in the parasite's genome could render this drug ineffective as have successively become all its predecessors throughout Pailin Given the impossibility of carrying out MSAT at the scale originally envisaged, mainly for reasons of lack of human resources in Cambodia, a strong case has been made in favour of Mass Drug Administration (MDA) throughout Pailin as a radical measure to rapidly eliminate In conclusion, FSAT may be a useful additional tool in the containment of AR parasites, among the other strategies implemented, albeit its shortcomings in terms of slowness and coverage in three important ways: (i) by providing information on travel and migrations patterns, (ii) by identifying clusters of asymptomatic carriers and (iii) by reducing the overall parasite biomass through the identification and treatment of infections that would otherwise have been undetected. However, recent a report demonstrating the emergence of AR malaria along the Myanmar-Thailand border emphasize the need to expand containment efforts and to further improve surveillance strategies Table S1P. falciparum bar-coding, Pailin, Cambodia, 2010.Sequences of primers used for (DOCX)Click here for additional data file.Table S2Clustering of asymptomatic carriers based on HC and VMW data in 2009 and 2010, Pailin, Cambodia(DOCX)Click here for additional data file.