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1.  Evaluating the feasibility of using insecticide quantification kits (IQK) for estimating cyanopyrethroid levels for indoor residual spraying in Vanuatu 
Malaria Journal  2014;13:178.
The quality of routine indoor residual spraying (IRS) operations is rarely assessed because of the limited choice of methods available for quantifying insecticide content in the field. This study, therefore, evaluated a user-friendly, rapid colorimetric assay for detecting insecticide content after routine IRS operations were conducted.
This study was conducted in Tafea Province, Vanuatu. Routine IRS was conducted with lambda cyhalothrin. Two methods were used to quantify the IRS activities: 1) pre-spray application of small felt pads and 2) post-spray removal of insecticide with adhesive. The insecticide content was quantified using a colorimetric assay (Insecticide Quantification Kit [IQK]), which involved exposing each sample to the test reagents for 15 mins. The concentration of insecticide was indicated by the depth of red colour.
The IQK proved simple to perform in the field and results could be immediately interpreted by the programme staff. The insecticide content was successfully sampled by attaching felt pads to the house walls prior to spraying. The IRS operation was well conducted, with 83% of houses being sprayed at the target dose (20 – 30 mg AI/m2). The average reading across all houses was 24.4 ± 1.5 mg AI/m2. The results from the felt pads applied pre-spray were used as a base to compare methods for sampling insecticide from walls post-spray. The adhesive of Sellotape did not collect adequate samples. However, the adhesive of the felt pads provided accurate samples of the insecticide content on walls.
The IQK colorimetric assay proved to be a useful tool that was simple to use under realistic field conditions. The assay provided rapid information on IRS spray dynamics and spray team performance, facilitating timely decision making and reporting for programme managers. The IQK colorimetric assay will have direct applications for routine quality control in malaria control programmes globally and has the potential to improve the efficacy of vector control operations.
PMCID: PMC4020589  PMID: 24885084
Colorimetric assay; Insecticide residual spray (IRS); Vanuatu; Insecticide quantification kit
2.  Plasmodium ovale: a case of not-so-benign tertian malaria 
Malaria Journal  2014;13:85.
Severe malaria is most commonly associated with Plasmodium falciparum. Plasmodium vivax is increasingly recognized as being capable of causing severe disease. In contrast, Plasmodium ovale is considered as a cause of benign disease and evidence supporting the occurrence of severe or complicated ovale infection is rare. This report describes a case of severe P. ovale infection in a patient presenting with jaundice, respiratory distress, severe thrombocytopenia, petechiae, and hypotension. He had no apparent underlying risk factors for severe disease.
PMCID: PMC3984724  PMID: 24612535
Plasmodium ovale; Severe; Malaria; Infection
3.  First case of a naturally acquired human infection with Plasmodium cynomolgi 
Malaria Journal  2014;13:68.
Since 1960, a total of seven species of monkey malaria have been reported as transmissible to man by mosquito bite: Plasmodium cynomolgi, Plasmodium brasilianum, Plasmodium eylesi, Plasmodium knowlesi, Plasmodium inui, Plasmodium schwetzi and Plasmodium simium. With the exception of P. knowlesi, none of the other species has been found to infect humans in nature. In this report, it is described the first known case of a naturally acquired P. cynomolgi malaria in humans.
The patient was a 39-year-old woman from a malaria-free area with no previous history of malaria or travel to endemic areas. Initially, malaria was diagnosed and identified as Plasmodium malariae/P. knowlesi by microscopy in the Terengganu State Health Department. Thick and thin blood films stained with 10% Giemsa were performed for microscopy examination. Molecular species identification was performed at the Institute for Medical Research (IMR, Malaysia) and in the Malaria & Emerging Parasitic Diseases Laboratory (MAPELAB, Spain) using different nested PCR methods.
Microscopic re-examination in the IMR showed characteristics of Plasmodium vivax and was confirmed by a nested PCR assay developed by Snounou et al. Instead, a different PCR assay plus sequencing performed at the MAPELAB confirmed that the patient was infected with P. cynomolgi and not with P. vivax.
This is the first report of human P. cynomolgi infection acquired in a natural way, but there might be more undiagnosed or misdiagnosed cases, since P. cynomolgi is morphologically indistinguishable from P. vivax, and one of the most used PCR methods for malaria infection detection may identify a P. cynomolgi infection as P. vivax.
Simian Plasmodium species may routinely infect humans in Southeast Asia. New diagnostic methods are necessary to distinguish between the human and monkey malaria species. Further epidemiological studies, incriminating also the mosquito vector(s), must be performed to know the relevance of cynomolgi malaria and its implication on human public health and in the control of human malaria.
The zoonotic malaria cannot be ignored in view of increasing interactions between man and wild animals in the process of urbanization.
PMCID: PMC3937822  PMID: 24564912
Plasmodium vivax; Plasmodium cynomolgi; Molecular methods; Malaysia; Simian malaria
4.  High prevalence of mutation in the Plasmodium falciparum dhfr and dhps genes in field isolates from Sabah, Northern Borneo 
Malaria Journal  2013;12:198.
Sulphadoxine-pyrimethamine (SP) has been in use for the treatment of uncomplicated falciparum malaria in Malaysia since the 1970s and is still widely employed in spite of widespread clinical resistance. Resistance to SP is known to be mediated by mutations in the pfdhfr and pfdhps genes. The aim of the present study was to investigate the distribution of pfdhfr and pfdhps gene polymorphism in Plasmodium falciparum field isolates from Kalabakan, Sabah, in northern Borneo.
A total number of 619 individuals were screened from 23 study sites of which 31 were positive for P. falciparum. Analysis of restriction fragment length polymorphisms (RFLP) was used to identify polymorphism in the pfdhfr and pfdhps genes at positions 16, 51, 59, 108, 164 and 437, 540, 581, respectively.
All samples had at least one mutation in each of the genes associated with drug resistance. The prevalence of pfdhfr 59arg, 164leu and 108asn were 100%, 80.65% and 58.06%, respectively. Pfdhps mutants 437gly and 581gly accounted for 100% and 74.19% respectively. In pfdhfr, the most common mutant genotypes were combination 59arg + 164leu (22.58%) and 59arg + 108asn + 164leu (51.61%). In pfdhps the most common genotype was 437gly + 581gly (74.19%). One individual (3.22%) harboured parasites with four pfdhfr (16 val + 59arg + 108asn + 164leu) and two pfdhps (437gly + 581gly) mutations. The highest quintuple pfdhfr/pfdhps (41.94%) was three pfdhfr (59arg + 108asn + 164gly) and two pfdhps (437gly + 581gly).
The data suggest a high prevalence of genetic variations conferring resistance to SP which can predict treatment failure before becoming clinically evident. In areas like this, the use of SP may no longer be indicated.
PMCID: PMC3706343  PMID: 23758930
Plasmodium falciparum; Sulphadoxine–pyrimethamine; Molecular markers
5.  Plasmodium vivax and Plasmodium falciparum infections in the Republic of Djibouti: evaluation of their prevalence and potential determinants 
Malaria Journal  2012;11:395.
Formerly known as a hypoendemic malaria country, the Republic of Djibouti declared the goal of pre-eliminating malaria in 2006. The aim of the present study was to evaluate the prevalence of Plasmodium falciparum, Plasmodium vivax and mixed infections in the Djiboutian population by using serological tools and to identify potential determinants of the disease and hotspots of malaria transmission within the country.
The prevalence of P. falciparum and P. vivax within the districts of the capital city and the rest of the Republic of Djibouti were assessed using 13 and 2 serological markers, respectively. The relationship between the immune humeral response to P. falciparum and P. vivax and variables such as age, gender, wealth status, urbanism, educational level, distance to rivers/lakes, living area, having fever in the last month, and staying in a malaria-endemic country more than one year was estimated and analysed by questionnaires administered to 1910 Djiboutians. Multivariate ordinal logistic regression models of the immune humeral response were obtained for P. falciparum and P. vivax.
The P. falciparum and P. vivax seroprevalence rates were 31.5%, CI95% [29.4-33.7] and 17.5%, CI95% [15.8-19.3], respectively. Protective effects against P. falciparum and P. vivax were female gender, educational level, and never having visited a malaria-endemic area for more than one year. For P. falciparum only, a protective effect was observed for not having a fever in the last month, living more than 1.5 km away from lakes and rivers, and younger ages.
This is the first study that assessed the seroprevalence of P. vivax in the Republic of Djibouti. It is necessary to improve knowledge of this pathogen in order to create an effective elimination programme. As supported by recent observations on the subject, the Republic of Djibouti has probably demonstrated a real decrease in the transmission of P. falciparum in the past seven years, which should encourage authorities to improve efforts toward elimination.
PMCID: PMC3544601  PMID: 23190709
Malaria; Plasmodium falciparum; Plasmodium vivax; Seroprevalence; Serological marker; Djibouti
7.  The baseline distribution of malaria in the initial phase of elimination in Sabang Municipality, Aceh Province, Indonesia 
Malaria Journal  2012;11:291.
Sabang Municipality, in Aceh Province, Indonesia, plans to initiate a malaria elimination programme in 2013. A baseline survey of the distribution of malaria in the municipality was conducted to lay the foundations for an evidence-based programme and to assess the island’s readiness to begin the elimination process.
The entire population of the municipality was screened for malaria infection and G6PD deficiency. Specimens collected included blood slides, blots and tubes for selected households.
Results and Discussion
Samples were collected from 16,229 residents. Microscopic examination of the blood smears revealed 12 malaria infections; 10 with Plasmodium falciparum and 2 with Plasmodium vivax. To confirm the parasite prevalence, polymerase chain reaction (PCR) diagnosis was performed on the entire positive cases by microscopy and randomized 10% of the microscopically negative blood samples. PCR revealed an additional 11 subjects with malaria; one P. falciparum infection from the village of Paya Keunekai, and nine P. vivax infections and one mixed P. falciparum/P. vivax infection from the village of Batee Shok. The overall slide positivity rate was 0.074% (CI 95%: 0.070 – 0.078) and PCR corrected prevalence 0,590% (CI 95%: 0.582 – 0.597). Analysis of 937 blood samples for G6PD deficiency revealed two subjects (0.2%) of deficient G6PD. Analysis of several genes of the parasite, such as Pfdhfr, Pfdhps, Pfmdr1, Pfcrt, Pfmsp1, Pfmsp2, Pvdhfr, Pvdhps, Pvmdr1 and host gene, such as G6PD gene revealed that both P. falciparum and P. vivax carried the mutation associated with chloroquine resistance.
Malariometric and host genetic analysis indicated that there is a low prevalence of both malaria and G6PD deficiency in the population of Sabang Municipality. Nevertheless, malaria cases were clustered in three rural villages and efforts for malaria elimination in Sabang should be particularly focused on those three villages.
PMCID: PMC3478225  PMID: 22908898
8.  Existence of the rdl mutant alleles among the anopheles malaria vector in Indonesia 
Malaria Journal  2012;11:57.
The gamma-aminobutyric acid (GABA) receptor-chloride channel complex is known to be the target site of dieldrin, a cyclodiene insecticide. GABA-receptors, with a naturally occurring amino acid substitution, A302S/G in the putative ion-channel lining region, confer resistance to cyclodiene insecticides that includes aldrin, chlordane, dieldrin, heptachlor, endrin and endosulphan.
A total of 154 mosquito samples from 10 provinces of malaria-endemic areas across Indonesia (Aceh, North Sumatra, Bangka Belitung, Lampung, Central Java, East Nusa Tenggara, West Nusa Tenggara, West Sulawesi, Molucca and North Molucca) were obtained and identified by species, using morphological characteristic. The DNA was individually extracted using chelex-ion exchanger and the DNA obtained was used for analyses using sequencing method.
Molecular analysis indicated 11% of the total 154 Anopheles samples examined, carried Rdl mutant alleles. All of the alleles were found in homozygous form. Rdl 302S allele was observed in Anopheles vagus (from Central Java, Lampung, and West Nusa Tenggara), Anopheles aconitus (from Central Java), Anopheles barbirostris (from Central Java and Lampung), Anopheles sundaicus (from North Sumatra and Lampung), Anopheles nigerrimus (from North Sumatra), whereas the 302 G allele was only found in Anopheles farauti from Molucca.
The existence of the Rdl mutant allele indicates that, either insecticide pressure on the Anopheles population in these areas might still be ongoing (though not directly associated with the malaria control programme) or that the mutant form of the Rdl allele is relatively stable in the absence of insecticide. Nonetheless, the finding suggests that integrated pest management is warranted in malaria-endemic areas where insecticides are widely used for other purposes.
PMCID: PMC3311089  PMID: 22364613
Anopheles; Diedrin; GABA; Receptor; Malaria; Rdl
9.  Mapping of mosquito breeding sites in malaria endemic areas in Pos Lenjang, Kuala Lipis, Pahang, Malaysia 
Malaria Journal  2011;10:361.
The application of the Geographic Information Systems (GIS) to the study of vector transmitted diseases considerably improves the management of the information obtained from the field survey and facilitates the study of the distribution patterns of the vector species.
As part of a study to assess remote sensing data as a tool for vector mapping, geographical features like rivers, small streams, forest, roads and residential area were digitized from the satellite images and overlaid with entomological data. Map of larval breeding habitats distribution and map of malaria transmission risk area were developed using a combination of field data, satellite image analysis and GIS technique. All digital data in the GIS were displayed in the WGS 1984 coordinate system. Six occasions of larval surveillance were also conducted to determine the species of mosquitoes, their characteristics and the abundance of habitats.
Larval survey studies showed that anopheline and culicine larvae were collected and mapped from 79 and 67 breeding sites respectively. Breeding habitats were located at 100-400 m from human settlement. Map of villages with 400 m buffer zone visualizes that more than 80% of Anopheles maculatus s.s. immature habitats were found within the buffer zone.
This study amplifies the need for a broadening of the GIS approach which is emphasized with the aim of rejuvenating the dynamic aspect of entomological studies in Malaysia. In fact, the use of such basic GIS platforms promote a more rational basis for strategic planning and management in the control of endemic diseases at the national level.
PMCID: PMC3265567  PMID: 22166101
10.  Molecular answers to the high failure rate of malaria RDTs 
Malaria Journal  2010;9(Suppl 2):P56.
PMCID: PMC2963299
11.  Seasonal distribution of anti-malarial drug resistance alleles on the island of Sumba, Indonesia 
Malaria Journal  2009;8:222.
Drug resistant malaria poses an increasing public health problem in Indonesia, especially eastern Indonesia, where malaria is highly endemic. Widespread chloroquine (CQ) resistance and increasing sulphadoxine-pyrimethamine (SP) resistance prompted Indonesia to adopt artemisinin-based combination therapy (ACT) as first-line therapy in 2004. To help develop a suitable malaria control programme in the district of West Sumba, the seasonal distribution of alleles known to be associated with resistance to CQ and SP among Plasmodium falciparum isolates from the region was investigated.
Plasmodium falciparum isolates were collected during malariometric surveys in the wet and dry seasons in 2007 using two-stage cluster sampling. Analysis of pfcrt, pfmdr1, pfmdr1 gene copy number, dhfr, and dhps genes were done using protocols described previously.
Results and Discussion
The 76T allele of the pfcrt gene is nearing fixation in this population. Pfmdr1 mutant alleles occurred in 72.8% and 53.3%, predominantly as 1042D and 86Y alleles that are mutually exclusive. The prevalence of amplified pfmdr1 was found 41.9% and 42.8% of isolates in the wet and dry seasons, respectively. The frequency of dhfr mutant alleles was much lower, either as a single 108N mutation or paired with 59R. The 437G allele was the only mutant dhps allele detected and it was only found during dry season.
The findings demonstrate a slighly higher distribution of drug-resistant alleles during the wet season and support the policy of replacing CQ with ACT in this area, but suggest that SP might still be effective either alone or in combination with other anti-malarials.
PMCID: PMC2761937  PMID: 19788738
12.  Health service providers in Somalia: their readiness to provide malaria case-management 
Malaria Journal  2009;8:100.
Studies have highlighted the inadequacies of the public health sector in sub-Saharan African countries in providing appropriate malaria case management. The readiness of the public health sector to provide malaria case-management in Somalia, a country where there has been no functioning central government for almost two decades, was investigated.
Three districts were purposively sampled in each of the two self-declared states of Puntland and Somaliland and the south-central region of Somalia, in April-November 2007. A survey and mapping of all public and private health service providers was undertaken. Information was recorded on services provided, types of anti-malarial drugs used and stock, numbers and qualifications of staff, sources of financial support and presence of malaria diagnostic services, new treatment guidelines and job aides for malaria case-management. All settlements were mapped and a semi-quantitative approach was used to estimate their population size. Distances from settlements to public health services were computed.
There were 45 public health facilities, 227 public health professionals, and 194 private pharmacies for approximately 0.6 million people in the three districts. The median distance to public health facilities was 6 km. 62.3% of public health facilities prescribed the nationally recommended anti-malarial drug and 37.7% prescribed chloroquine as first-line therapy. 66.7% of public facilities did not have in stock the recommended first-line malaria therapy. Diagnosis of malaria using rapid diagnostic tests (RDT) or microscopy was performed routinely in over 90% of the recommended public facilities but only 50% of these had RDT in stock at the time of survey. National treatment guidelines were available in 31.3% of public health facilities recommended by the national strategy. Only 8.8% of the private pharmacies prescribed artesunate plus sulphadoxine/pyrimethamine, while 53.1% prescribed chloroquine as first-line therapy. 31.4% of private pharmacies also provided malaria diagnosis using RDT or microscopy.
Geographic access to public health sector is relatively low and there were major shortages of appropriate guidelines, anti-malarials and diagnostic tests required for appropriate malaria case management. Efforts to strengthen the readiness of the health sector in Somalia to provide malaria case management should improve availability of drugs and diagnostic kits; provide appropriate information and training; and engage and regulate the private sector to scale up malaria control.
PMCID: PMC2688519  PMID: 19439097
13.  Seasonal prevalence of malaria in West Sumba district, Indonesia 
Malaria Journal  2009;8:8.
Accurate information about the burden of malaria infection at the district or provincial level is required both to plan and assess local malaria control efforts. Although many studies of malaria epidemiology, immunology, and drug resistance have been conducted at many sites in Indonesia, there is little published literature describing malaria prevalence at the district, provincial, or national level.
Two stage cluster sampling malaria prevalence surveys were conducted in the wet season and dry season across West Sumba, Nusa Tenggara Province, Indonesia.
Eight thousand eight hundred seventy samples were collected from 45 sub-villages in the surveys. The overall prevalence of malaria infection in the West Sumba District was 6.83% (95% CI, 4.40, 9.26) in the wet season and 4.95% (95% CI, 3.01, 6.90) in the dry. In the wet season Plasmodium falciparum accounted for 70% of infections; in the dry season P. falciparum and Plasmodium vivax were present in equal proportion. Malaria prevalence varied substantially across the district; prevalences in individual sub-villages ranged from 0–34%. The greatest malaria prevalence was in children and teenagers; the geometric mean parasitaemia in infected individuals decreased with age. Malaria infection was clearly associated with decreased haemoglobin concentration in children under 10 years of age, but it is not clear whether this association is causal.
Malaria is hypoendemic to mesoendemic in West Sumba, Indonesia. The age distribution of parasitaemia suggests that transmission has been stable enough to induce some clinical immunity. These prevalence data will aid the design of future malaria control efforts and will serve as a baseline against which the results of current and future control efforts can be assessed.
PMCID: PMC2628667  PMID: 19134197

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