Spatial and temporal variations of malaria epidemic risk in Ethiopia: factors involved and implications

The aim of this study was to describe spatial and temporal variations in malaria epidemic risk in Ethiopia and to examine factors involved in relation to their implications for early warning and interpretation of geographical risk models. Forty-eight epidemic episodes were identified in various areas between September 1986 and August 1993 and factors that might have led to the events investigated using health facility records and weather data. The study showed that epidemics in specific years were associated with specific geographical areas. A major epidemic in 1988 affected the highlands whereas epidemics in 1991 and 1992 affected highland-fringe areas on the escarpments of the Rift Valley and in southern and north-western parts of the country. Malaria epidemics were significantly more often preceded by a month of abnormally high minimum temperature in the preceding 3 months than based on random chance, whereas frequency of abnormally low minimum temperature prior to epidemics was significantly lower than expected. Abnormal increases of maximum temperature and rainfall had no positive association with the epidemics. A period of low incidence during previous transmission seasons might have aggravated the events, possibly due to low level of immunity in affected populations. Epidemic risk is a dynamic phenomenon with changing geographic pattern based on temporal variations in determinant factors including weather and other eco-epidemiological characteristics of areas at risk. Epidemic early warning systems should take account of non-uniform effects of these factors by space and time and thus temporal dimensions need to be considered in spatial models of epidemic risks.     

A preliminary study on pro- and anti-inflammatory cytokine profiles in Plasmodium vivax malaria patients from central zone of India

The aim of this preliminary study was to investigate the plasma cytokine profiles in a group of patients suffering from Plasmodium vivax malaria during the peak of its transmission season. Plasma samples of 173 P. vivax patients and 34 healthy individuals were analyzed for IFN-γ, TNF-α, IL-10 and IP-10 levels by ELISA. Levels of both pro- and anti-inflammatory cytokines were significantly higher in P. vivax patients compared to controls. Children with P. vivax infection had significantly higher levels of IFN-γ than adults (P = 0.017). Asexual parasitaemia versus TNF-α (r = −0.31, P = 0.01), IL-10 (r = −0.30, P = 0.015) and gametocytaemia versus IFN-γ (r = −0.26; P = 0.034) levels showed significant negative correlation in children compared to adults. The median concentrations of IFN-γ (P = 0.001), IL-10 (P = 0.032) and IP-10 (P ≤ 0.05) were higher in children reported with chills and rigors, whereas in adults only IFN-γ levels was higher (P < 0.0001). The median plasma concentrations of IFN- γ (P = 0.02), IL-10 (P < 0.0001) and IP-10 (P = 0.068) were higher in patients with mild anaemia compared to non-anaemic patients. The results indicated that both pro- and anti-inflammatory cytokine responses are associated with clinical signs of mild anaemia and paroxysm during symptomatic P. vivax malaria in Central India.     

Substantial increase of malaria in inland areas of eastern French Guiana

This study includes malaria cases diagnosed by the Parasitology Unit of the Cayenne Hospital (French Guiana) using the same procedure from 1996 to 2003. We provide data for two main rural communities in slightly inland areas of eastern French Guiana (Cacao, Regina) and for Cayenne, the capital of this French department. The frequency of bouts of malaria has been increasing rapidly since mid-2001, in these regions that were virtually considered to be malaria-free. This substantial increase of malaria appears mainly to involve Plasmodium vivax and recent Brazilian immigrants. Other plausible explanations which could account for the observed trend are discussed.     

Detection of Plasmodium vivax and Plasmodium falciparum DNA in human saliva and urine: Loop-mediated isothermal amplification for malaria diagnosis

This study investigated loop-mediated isothermal amplification (LAMP) detection of Plasmodium falciparum and Plasmodium vivax in urine and saliva of malaria patients. From May to November 2011, 108 febrile patients referred to health centers in Sistan and Baluchestan Province of south-eastern Iran participated in the study. Saliva, urine, and blood samples were analyzed with nested PCR and LAMP targeting the species-specific nucleotide sequence of small subunit ribosomal RNA gene (18S rRNA) of P. falciparum and P. vivax and evaluated for diagnostic accuracy by comparison to blood nested PCR assay. When nested PCR of blood is used as standard, microscopy and nested PCR of saliva and urine samples showed sensitivity of 97.2%, 89.4% and 71% and specificity of 100%, 97.3% and 100%, respectively. LAMP sensitivity of blood, saliva, and urine was 95.8%, 47% and 29%, respectively, whereas LAMP specificity of these samples was 100%. Microscopy and nested PCR of saliva and LAMP of blood were comparable to nested PCR of blood (к = 0.95, 0.83, and 0.94, respectively), but agreement for nested PCR of urine was moderate (к = 0.64) and poor to fair for saliva LAMP and urine LAMP (к = 0.38 and 0.23, respectively). LAMP assay showed low sensitivity for detection of Plasmodium DNA in human saliva and urine compared to results with blood and to nested PCR of blood, saliva, and urine. However, considering the advantages of LAMP technology and of saliva and urine sampling, further research into the method is worthwhile. LAMP protocol and precise preparation protocols need to be defined and optimized for template DNA of saliva and urine.     

Idiopathic thrombocytopenic purpura due to vivax malaria in the Brazilian Amazon

We describe a rare case of idiopathic thrombocytopenic purpura (ITP) triggered by Plasmodium vivax infection. The patient developed thrombocytopenia and bleeding associated with three episodes of malaria, and became dependent on corticosteroid therapy. The mechanisms by which this parasite evokes thrombocytopenia remain obscure.     

Public health impact of drug resistant Plasmodium falciparum malaria

The alarming increase in Plasmodium falciparum resistance to commonly used anti-malarial drugs represents a major public health threat. The impact is however difficult to quantify. In low transmission areas, an increase in acute manifestations ("epidemic") is often quickly apparent and resistance is rapidly propagated due to high drug pressure on existing parasite populations. In high transmission areas, the clinical effects are mainly prolonged/chronic infections with increasing risk of severe anemia. Mortality estimates from public health records in Africa generally suggest significant increases (from 2- to 11-fold) in malaria-associated mortality among children when resistance develops and spreads. Hospital attendances and admissions show similar trends. Change of policy to alternative efficacious treatment with radical cure is necessary at an earlier stage (from 10% treatment failure) than previously assumed in order to prevent deaths in millions of African children. Early switch to artemisinin based combination therapy (ACT) represents such a critical and urgent strategy.     

Changing patterns of forest malaria among the mobile adult male population in Chumkiri District, Cambodia

Forest malaria remains a major problem in many parts of Southeast Asia and South America. In Cambodia, where a significant reduction of malaria morbidity and mortality has been observed in the last 20 years, the forest malaria situation was studied in Chumkiri District by analysing the available passive case detection data and conducting malariometric (n=1018) and questionnaire surveys (n=374) in four forest-fringe villages. There has been a decreasing trend of malaria incidence from 2001. Plasmodium falciparum was highly predominant and P. vivax was rare. The nearby-forest villages showed significantly higher parasite rates than the far-from-forest villages (9.0% vs. 1.2%, p<0.01). Malaria was highly restricted to the male adults but was nearly non-existent in other accompanying family members, including small children and females. Low income and working in forests were strongly associated with the malaria risk. Our results suggest that transmission has greatly reduced in forest-fringe villages, but remains active in forests, which is primarily maintained between the forest vector Anopheles dirus and ethnic minority inhabitants. Specific interventions directed to these previously neglected in-forest inhabitants to protect themselves and male adult villagers during their forest activities are necessary to achieve an ultimate goal of malaria elimination from Cambodia.     

Genetic diversity of the msp-1 locus and symptomatic malaria in south-west Nigeria

Genetic characteristics of Plasmodium falciparum may play a role in the clinical severity of malaria infection. We have studied the association between diversity at the merozoite surface protein-1 (msp-1) locus and the severity of disease in childhood malaria in Ibadan, south-west Nigeria. Two hundred and twenty-three children (median age of 34.5 months) presenting with malaria were enrolled into the study. They comprised 53 children with asymptomatic malaria (ASM), 101 with acute uncomplicated malaria (UM) and 69 with severe malaria (SM). Genotyping of the msp-1 locus was by polymerase chain reaction. The distribution of msp-1 alleles was significantly different between the three groups. Asymptomatic malaria samples had a higher median number of alleles than the other two groups. The type of msp-1 allele detected was significantly associated with the clinical category of malaria. The absence of K1 alleles was associated with a three-fold increase risk of UM and a four-fold increased risk of SM when compared with asymptomatic malaria. The absence of MAD20 alleles was associated with a five-fold increase risk of UM and an eight-fold increase of SM. We have found an association between the msp-1 locus of P. falciparum and clinical severity of malaria in a sample of Nigerian children. Our findings show that the presence of the K1 and MAD20 alleles was significantly associated with ASM and consequently a reduced risk of developing the symptomatic disease.     

Plasma and in vitro levels of cytokines during and after a Plasmodium falciparum malaria attack in Gabon

Fifty subjects living in a malaria endemic area were studied at diagnosis of a Plasmodium falciparum attack and 3 weeks later. Absolute numbers of CD3(+), CD4(+) and CD8(+) lymphocytes as well as plasma cytokines and secreted cytokines after in vitro mitogenic stimulation were measured. At enrollment, lymphopenia was observed, lending support to the reallocation hypothesis during the acute phase. A significant elevation of the number of CD8(+) cells was present in the peripheral blood during the recovery phase. During the acute phase, plasma IL-6 levels peaked while in vitro production capacity was high at both phases. Plasma IL-6 concentrations were positively related to blood parasite density at D0, as IL-4 and IFN-gamma, suggesting an early intervention of these cytokines. Plasma IL-2 levels were low at diagnosis although cells retained their ability to produce IL-2, which was found more frequently in plasma after cure. Acquisition of immunity with age was in relation with greater secretion abilities of cells for type 1 and type 2 cytokines during the parasite clearance phase. We conclude to an early implication of type 2 cytokines and IFN-gamma, with particularly high levels of IL-6.     

Naturally acquired immunity to Plasmodium falciparum malaria in Africa

Infection by Plasmodium falciparum parasites can lead to substantial protective immunity to malaria, and available evidence suggest that acquisition of protection against some severe malaria syndromes can be fairly rapid. Although these facts have raised hopes that the development of effective vaccines against this major cause of human misery is a realistic goal, the uncertainty regarding the antigenic targets of naturally acquired protective immunity and the immunological mechanisms involved remain major vaccine development obstacles. Nevertheless, a coherent theoretical framework of how protective immunity to P. falciparum malaria is acquired following natural exposure to the parasites is beginning to emerge, not least thanks to studies that have combined clinical and epidemiological data with basic immunological research. This framework involves IgG with specificity for clonally variant antigens on the surface of the infected erythrocytes, can explain some of the difficulties in relating particular immune responses with specificity for well-defined antigenic targets to clinical protection, and suggests a radically new approach to controlling malaria-related morbidity and mortality by immunological means.     

Anthropophilic mosquitoes and malaria transmission in the eastern foothills of the central highlands of Madagascar

Malaria remains a major public health problem in Madagascar, as it is the first cause of morbidity in health care facilities. Its transmission remains poorly documented. An entomological study was carried out over 1 year (October 2003-September 2004) in Saharevo, a village located at an altitude of 900 m on the eastern edge of the Malagasy central highlands. Mosquitoes were sampled weekly upon landing on human volunteers and in various resting-places. Out of 5515 mosquitoes collected on humans, 3219 (58.4%) were anophelines. Eleven anopheline species were represented, among which Anopheles funestus, Anopheles gambiae, Anopheles arabiensis and Anopheles mascarensis. Out of 677 mosquitoes collected in bedrooms by pyrethrum spray catches and in Muirhead-Thomson pits, 656 (96.9%) were anopheline belonging to these four latter species. The proportion of mosquitoes that fed on human varied according to the resting-places and the mosquito species: 86% of An. funestus resting in bedrooms fed on humans, whereas only 16% of An. funestus and 0% of An. mascarensis resting in pits fed on humans. The proportion of anopheline mosquitoes infected with human Plasmodium was measured by circumsporozoite protein-ELISA: 10/633 An. funestus (1.58%), 1/211 An. gambiae s.l. (0.48%) and 2/268 An. mascarensis (0.75%). The annual entomological inoculation rate (number of bites of infected anophelines per adult) was estimated at 2.78. The transmission was mainly due to An. funestus and only observed in the second half of the rainy season, from February to May. These results are discussed in the context of the current malaria vector control policy in Madagascar.     

Epidemiological situation of malaria in Madagascar: Baseline data for monitoring the impact of malaria control programmes using serological markers

The aim of this study was to provide baseline information of the epidemiological situation of malaria in Madagascar using serological markers. We carried out cross-sectional studies in schoolchildren from eight sites in the four different malarious epidemiological strata of Madagascar. We studied the prevalence of anti-MSP1 antibodies to assess the burden, and anti-CSP antibodies to estimate the transmission intensity, of malaria. The overall prevalence of each antibody tested was 46.1% for anti-PfMSP-1, 15.2% for anti-PvMSP-1, 14.9% for anti-PfCSP, 4.9% for anti-PvCSP and 2.4% for anti-PmCSP. The prevalence of the five antibodies varied significantly between the sites (P < 10⁻⁶). We also found significant effects of ethnic origin on the prevalence of anti-PfMSP1 antibodies. With regular testing in the same target populations, this data will be particularly useful for managing the elimination strategy supported by the Malagasy Government.     

Relation between vitamin B12 and folate status, and hemoglobin concentration and parasitemia during acute malaria infections in Colombia

Anemia is a common complication of human malaria. Since micronutrient deficiencies are highly prevalent in malaria-endemic areas and appear to contribute to anemia etiology, we conducted a cross-sectional study in Tumaco, Colombia, to examine the associations between plasma vitamin B12 or erythrocyte folate concentrations and hemoglobin (Hb) among 96 adults with predominantly Plasmodium falciparum malaria. Prevalence of folate and vitamin B12 deficiencies was 26.0 and 26.6%, respectively. There was an inverse, linear relation between folate and Hb concentrations. Adjusted difference in Hb between lowest and highest folate quartiles was 1 g/dL (p = 0.04; p, test for trend = 0.01). Vitamin B12 was not associated with Hb concentrations and did not modify the associations between folate and Hb. Incidentally, body mass index (BMI) was inversely associated with parasitemia and risk of clinical malaria. Future longitudinal studies are warranted to determine the potential pathophysiological role of folate in malaria-related anemia.     

A new visually improved and sensitive loop mediated isothermal amplification (LAMP) for diagnosis of symptomatic falciparum malaria

Molecular diagnosis of malaria by nucleotide amplification requires sophisticated and expensive instruments, typically found only in well-established laboratories. Loop-mediated isothermal amplification (LAMP) has provided a new platform for an easily adaptable molecular technique for molecular diagnosis of malaria without the use of expensive instruments. A new primer set has been designed targeting the 18S rRNA gene for the detection of Plasmodium falciparum in whole blood samples. The efficacy of LAMP using the new primer set was assessed in this study in comparison to that of a previously described set of LAMP primers as well as with microscopy and real-time PCR as reference methods for detecting P. falciparum. Pre-addition of hydroxy napthol blue (HNB) in the LAMP reaction caused a distinct color change, thereby improving the visual detection system. The new LAMP assay was found to be 99.1% sensitive compared to microscopy and 98.1% when compared to real-time PCR. Meanwhile, its specificity was 99% and 100% in contrast to microscopy and real-time PCR, respectively. Moreover, the LAMP method was in very good agreement with microscopy and real-time PCR (0.94 and 0.98, respectively). This new LAMP method can detect at least 5 parasites/μL of infected blood within 35 min, while the other LAMP method tested in this study, could detect a minimum of 100 parasites/μL of human blood after 60 min of amplification. Thus, the new method is sensitive and specific, can be carried out in a very short time, and can substitute PCR in healthcare clinics and standard laboratories.     

Genetic structure of Plasmodium vivax isolates from two malaria endemic areas in Afghanistan

In this study, the nature and extent of genetic diversity of Plasmodium vivax populations circulating in Afghanistan have been investigated by analyzing three genetic markers: csp, msp-1, and msp-3α. Blood samples (n = 202) were collected from patients presenting with vivax malaria from south-western (Herat) and south-eastern (Nangarhar) parts of Afghanistan, and analysed using nested-PCR/RFLP and sequencing methods. Genotyping pvmsp-1 revealed type 1, type 2 and recombinant type 3 allelic variants, with type 1 predominant in parasites in both study areas. The sequence analysis of 57 P. vivax isolates identified a total of 26 distinct alleles. Genotyping pvcsp gene showed that VK210 type (86.6%) is predominant in Afghanistan. Moreover, three major types of the pvmsp-3α locus: type A, type B and type C were distinguished among Afghani isolates. The predominant fragments among Nangarhar and Herat parasites were type A (70.8% and 67.9%, respectively). PCR/RFLP products with Hha I and Alu I were detected 52 and 38 distinct variants among Nangarhar and Herat isolates, respectively. These results strongly indicate that the P. vivax populations in Afghanistan are highly diverse.     

Relationship between entomological inoculation rate,Plasmodium falciparum prevalence rate,and incidence of malaria attack in rural Gabon

To assess the relationships between variations of Plasmodium falciparum transmission and those of peripheral parasitaemia prevalence or malaria attack incidence rates in regions with limited fluctuations of transmission, we conducted a follow-up in two Gabonese populations. Entomological surveys were carried out from May 1995 to April 1996 in Dienga, and from May 1998 to April 1999 in Benguia. In Dienga, malaria transmission was seasonal, being not detected during two 3-month periods. Mean entomological inoculation rate (EIR) was 0.28 infective bite/person/night. In Benguia, malaria transmission was perennial with seasonal fluctuations, mean EIR being 0.76 infective bite/person/night. In Dienga, 301 schoolchildren were followed from October 1995 to March 1996. Clinical malaria attack was defined as fever associated with >5000 parasites/microl of blood. P. falciparum prevalence varied from 28 to 42%, and monthly malaria attack incidence from 30 to 169 per thousand. In Benguia, the entire population (122 persons) was followed from November 1998 to April 1999. Prevalence varied from 22 to 50%, and monthly malaria attack incidence from 52 to 179 per thousand. In each area, entomological variations were not related to parasite prevalence, but preceded malaria attack incidence with 1- or 2-month time lag, corresponding to the pre-patency period that differs in the two populations, possibly according to differences in immunity related to parasite transmission.     

Anemia and malaria at different altitudes in the western highlands of Kenya

Malaria associated severe anemia in children is the most important complication of Plasmodium falciparum infection in sub-Saharan Africa. To evaluate anemia and malaria in an area with recurrent malaria epidemics in the western highlands of Kenya, we conducted cross-sectional surveys in four "lowland" (1440-1660 m) and two "highland" (1960 and 2040 m) villages in 2002. Among 1314 subjects randomly selected from all age groups, the overall prevalence of anemia (hemoglobin, Hb < 11 g/dl) was 14% and P. falciparum infection 17%. In children < or =5 years, anemia prevalence ranged from 57% at 1440 m to 11% at 2040 m and correlated with altitude (r = -0.88, P < 0.05). Similarly, P. falciparum prevalence ranged from 31 to 0% and correlated with altitude (r = -0.93, P < 0.01). Malnutrition defined by a body mass index <15th percentile characterized 39% of the population and the hookworm prevalence was 3.9%. In the lowland villages, anemia was most common in children < or =5 years of age (34%) followed by women of childbearing age (16%). A similar pattern was also observed in the highland villages. In these vulnerable populations, hemoglobin concentration was significantly associated with malaria infection, but not with malnutrition or hookworm infestation and comparisons of anemia prevalence between highland and lowland villages revealed that two-thirds of anemia could be attributed to malaria infection. The prevalence of severe anemia (Hb < 8 g/dl) was 1.5%; of these, 90% resided in lowland villages, 70% were under-fives, while 20% were women of childbearing age. In severely anemic subjects, the Hb concentration decreased further with malnutrition (P < 0.05). Anemia was more prevalent in the lowland villages characterized by high prevalence of P. falciparum infection. We conclude that malaria may also be the main cause of anemia in the highland fringe areas of sub-Saharan Africa. Measures that reduce the prevalence of malaria will consequently reduce anemia in both, young children and adult women and the need for blood transfusions associated with the risk of HIV-transmission.     

Some aspects of the behavior of the hypothalamus-pituitary-adrenal axis in patients with uncomplicated Plasmodium falciparum malaria: Cortisol and dehydroepiandrosterone levels

We studied the behavior of cortisol and dehydroepiandrosterone (DHEA) in 24 patients with uncomplicated Plasmodium falciparum malaria of the Evandro Chagas Institute, Belém, Pará, Brazil. The patients were evaluated before treatment (Day 0), 24h after the beginning of medication (Day 1) and on Day 8 of follow-up (Day 7). Steroid levels were correlated with parasitemia, temperature and time of the disease. The levels of these hormones were found to be significantly higher on Day 0 than on Day 7, showing no correlation with parasitemia or temperature, but temperature had a positive effect on the correlation between cortisol and dehydroepiandrosterone. Cortisol was not correlated with the time of disease, but a significant negative correlation was observed between DHEA and time of disease on Day 7, suggesting a decline in the adrenal reserve of this steroid. In conclusion, an increase in cortisol and dehydroepiandrosterone is observed in patients with falciparum malaria, with these levels declining with decreasing parasitemia. The finding that temperature interfered with the correlation between cortisol and dehydroepiandrosterone suggests a common mechanism for the activation of these hormones in malaria.     

High prevalence of PfCRT K76T mutation in Plasmodium falciparum isolates in Ghana

Plasmodium falciparum has successfully developed resistance to almost all currently used antimalarials. A single nucleotide polymorphism in the P. falciparum chloroquine resistance transporter (Pfcrt) gene at position 76 resulting in a change in coding from lysine to threonine (K76T) has been implicated to be the corner stone of chloroquine resistance. Widespread resistance to chloroquine in endemic regions led to its replacement with other antimalarials. In some areas this replacement resulted in a reversion of the mutant T76 allele to the wild-type K76 allele. This study was conducted to determine the prevalence of the K76T mutation of the Pfcrt gene eight years after the ban on chloroquine sales and use. A cross-sectional study was conducted in 6 regional hospitals in Ghana. PCR-RFLP was used to analyse samples collected to determine the prevalence of Pfcrt K76T mutation. Of the 1318 participants recruited for this study, 246 were found to harbour the P. falciparum parasites, of which 60.98% (150/246) showed symptoms for malaria. The prevalence of the Pfcrt T76 mutant allele was 58.54% (144/246) and that of the K76 wild-type allele was 41.46% (102/246). No difference of statistical significance was observed in the distribution of the alleles in the symptomatic and asymptomatic participants (P = 0.632). No significant association was, again, observed between the alleles and parasite density (P = 0.314), as well as between the alleles and Hb levels of the participants (P = 0.254). Notwithstanding the decline in the prevalence of the Pfcrt T76 mutation since the antimalarial policy change in 2004, the 58.54% prevalence recorded in this study is considered high after eight years of the abolishment of chloroquine usage in Ghana. This is in contrast to findings from other endemic areas where the mutant allele significantly reduced in the population after a reduction chloroquine use.     

Molecular typing of Plasmodium falciparum from Giemsa-stained blood smears confirms nosocomial malaria transmission

In this report, we describe the partial molecular characterisation of Plasmodium falciparum isolates obtained from two individuals who were involved in a probable case of accidental malaria transmission after admission to a hospital in the metropolitan area of S?o Paulo, Brazil. Molecular analysis of polymorphic stretches of the merozoite surface protein 1 and 2 genes using PCR-typing and nucleotide sequencing revealed that the two isolates were identical and that the identified msp-1 gene was different from all others published to date. Additional anamnestic data supported our findings and made all other possible routes of infection unlikely. The methodology used here is simple to perform and needs as little as one Giemsa-stained blood smear as starting material.