Social Sciences and Malaria Control
Trop Med Int Health 2003 Apr;8(4):290-296
Malaria morbidity, treatment-seeking behaviour, and mortality in a cohort of
young children in rural Burkina Faso.
Muller O, Traore C, Becher H, Kouyate B.
Department of Tropical Hygiene and Public Health of the Ruprecht-Karls-University,
Heidelberg, Germany Centre de Recherche en Sante de Nouna, Nouna, Burkina Faso.
OBJECTIVE: To describe the pattern of fever-associated morbidity,
treatment-seeking behaviour for fever episodes, and cause-specific mortality in
young children of a malaria-holoendemic area in rural Burkina Faso. METHODS: In
a longitudinal community-based intervention study, 709 representative children
aged 6-31 months were followed daily over 6 months (including the main malaria
transmission period) through village-based field staff. RESULTS: Of 1848 disease
episodes, 1640 (89%) were fever episodes, and of those, 894 (55%) were
attributed to malaria (fever + >/=5000 parasites/&mgr;l). Eighty-five percent of
fever episodes were treated, mainly with chloroquine and paracetamol, 69% of
treatments took place in households, 16% in local health centres, 13% in
villages, and 1% in hospitals. Treatment-seeking in a health centre or hospital
was associated with accessibility and disease severity. Cerebral malaria and
malnutrition-associated diarrhoea were the most frequently diagnosed causes of
death. While most children with a post-mortem diagnosis of diarrhoea had not
received any treatment, children who died of malaria had often received
insufficient treatment. In particular, there was a lack of an appropriate
second-line treatment at formal health services after chloroquine treatment had
failed to resolve symptoms. CONCLUSIONS: These findings call for more effective
prevention and treatment of malaria, malnutrition and diarrhoea in rural African
communities, as well as for better supervision of existing malaria treatment
guidelines in formal health services. PMID: 12667146
DISSERTATION ABSTRACTS INTERNATIONAL. VOLUME 63/07-A OF PAGE 2705.
Spatial configuration of malaria risk on the Amazon frontier: The hidden
reality behind global analysis (Brazil). 2002. 293 pages. (Dissertation)
Castro, Marcia Caldas de. Princeton University.
This dissertation investigates the interrelationships between macro-political, social, and economic polices, human migration, agricultural development, and malaria transmission on the Brazilian Amazon frontier. The ultimate goals are to: (i) identify the local determinants of malaria transmission, and thereby provide the necessary information for the design of more efficient malaria mitigation policies; and (ii) establish a spatially explicit methodology for analysis of malaria risk in complex ecosystems on the Amazon frontier. The analysis is focused on the Machadinho Settlement Project, located in the state of Rondônia. Field survey data collected in 1985 (the inception of the settlement), 1986, 1987 and 1995, provide information on health, demographic, economic, social, ecological, behavioral, and agricultural characteristics. I adopted a three-step methodological approach that combined spatial analysis, geostatistical tools, and fuzzy-set models. This innovative approach facilitated the analysis of malaria risk factors at a local level. Applying Grade of Membership model obtained nuanced profiles of low and high malaria risk for each of the four years studied, and for different levels of spatial aggregation. The results highlighted major variation over time and space, and characterized the subtle interface between natural and man-made environments for malaria transmission. They also showed that, as the spatial level of aggregation was refined, more detailed risk profiles were obtained. The risk profile representations facilitated the specification of a set of strategies that should be implemented in colonization projects, targeted in both time and space, for increasing the effectiveness and reducing the costs of malaria mitigation in frontier settlements. Important features of the proposed mitigation strategies include: (i) all persons arriving at new settlement projects should be tested and treated for malaria before they occupy their plots; (ii) environmental management should be a central focus in the early stages of settlement; (iii) personal behavior should be the focus of interventions after there is substantial land clearance and stabilization of the area; (iv) financial support for house construction must be guaranteed; (v) education about malaria should be provided to all settlers in the early stages of the project; (vi) promotion of a diverse array of supportive community organizations is essential.
BMC Int Health Hum Rights 2003 Apr 1;3(1):2
Global plagues and the Global Fund: Challenges in the fight against HIV,
TB and malaria.
Tan D, Upshur RE, Ford N.
Background: Although a grossly disproportionate burden of disease from HIV/AIDS,
TB and malaria remains in the Global South, these infectious diseases have
finally risen to the top of the international agenda in recent years. Ideal
strategies for combating these diseases must balance the advantages and
disadvantages of 'vertical' disease control programs and 'horizontal'
capacity-building approaches. Discussion: The Global Fund to Fight AIDS,
Tuberculosis and Malaria (GFATM) represents an important step forward in the
struggle against these pathogens. While its goals are laudable, significant
barriers persist. Most significant is the pitiful lack of funds committed by
world governments, particularly those of the very G8 countries whose discussions
gave rise to the Fund. A drastic scaling up of resources is the first clear
requirement for the GFATM to live up to the international community's lofty
intentions. A directly related issue is that of maintaining a strong commitment
to the treatment of the three diseases along with traditional prevention
approaches, with the ensuing debates over providing affordable access to
medications in the face of the pharmaceutical industry's vigorous protection of
patent rights. Summary: At this early point in the Fund's history, it remains to
be seen how these issues will be resolved at the programming level.
Nevertheless, it is clear that significant structural changes are required in
such domains as global spending priorities, debt relief, trade policy, and
corporate responsibility. HIV/AIDS, tuberculosis and malaria are global problems
borne of gross socioeconomic inequality, and their solutions require
correspondingly geopolitical solutions. PMID: 12667262
Biol Chem 2003 Jan;384(1):71-82
MSP-1 malaria pseudopeptide analogs: biological and immunological significance
and three-dimensional structure.
Lozano JM, Alba MP, Vanegas M, Silva Y, Torres-Castellanos JL, Patarroyo ME.
Fundacion Instituto de Inmunologia de Colombia, Carrera 50 No. 26-00, Bogota,
Merozoite Surface Protein-1 (MSP-1) has been considered as a malaria vaccine
candidate. It is processed during the Plasmodium falciparum invasion process of
red blood cells (RBCs). A conserved MSP-1 C-terminal peptide was identified as a
high-activity erythrocyte-binding peptide (HAEBP) termed 1585. Since conserved
HAEBPs are neither antigenic nor immunogenic we decided to assess the
significance of a single peptide bond replacement in 1585. Thus, two
pseudopeptides were obtained by introducing a Y[CH2-NH] reduced amide isoster
into the 1585 critical binding motif. The pseudopeptides bound to different
HLA-DR alleles, suggesting that backbone modifications affect MHC-II binding
patterns. Pseudopeptide-antibodies inhibit in vitro parasite RBC invasion by
recognizing MSP-1. Each pseudopeptide-induced antibody shows distinct
recognition patterns. 1H-NMR studies demonstrated that isoster bonds modulate
the pseudopeptides' structure and thus their immunological properties, therefore
representing a possible subunit malaria vaccine component. PMID: 12674501
Proc Natl Acad Sci U S A 2003 Apr 2;
Erythrocyte-binding antigen 175 mediates invasion in Plasmodium falciparum
utilizing sialic acid-dependent and -independent pathways.
Duraisingh MT, Maier AG, Triglia T, Cowman AF.
The Walter and Eliza Hall Institute of Medical Research, Melbourne 3050,
The Plasmodium falciparum erythrocyte-binding antigen 175 (EBA-175) is a ligand
for merozoite invasion into human erythrocytes that binds to glycophorin A in a
sialic acid-dependent manner. P. falciparum strain W2mef depends on sialic acid
for invasion of erythrocytes, whereas 3D7 is sialic acid-independent. We
generated parasites that lack expression or express truncated forms of EBA-175
in W2mef and 3D7. Lack of EBA-175 expression in W2mef parasites was associated
with a switch to sialic acid-independent invasion. 3D7 parasites lacking
expression of EBA-175 showed no alteration in their ability to utilize sialic
acid-independent pathways. Strikingly, both W2mef and 3D7 parasites lacking
EBA-175 expression invaded chymotrypsin-treated erythrocytes inefficiently
compared with the parental lines. This loss of function suggests that the
EBA-175/glycophorin A ligand-receptor interaction is the major
chymotrypsin-resistant invasion pathway. Parasite lines with truncated EBA-175
had invasion phenotypes equivalent to parasites lacking expression of EBA-175.
The EBA-175 ligand is functional in erythrocyte invasion by merozoites that
utilize either sialic acid-dependent or -independent invasion pathways. This
finding suggests a model where a minimal affinity supplied by multiple
ligand-receptor interactions is required for successful invasion and has
implications for EBA-175 as a malaria vaccine candidate. PMID: 12672957
Mol Biochem Parasitol 2003 Apr;127(2):179-91
The 3D7var5.2 (var(COMMON)) type var gene family is commonly expressed
in non-placental Plasmodium falciparum malaria.
Winter G, Chen Q, Flick K, Kremsner P, Fernandez V, Wahlgren M.
Microbiology and Tumor Biology Center, Karolinska Institutet, P.O. Box 280,
SE-171 77, Stockholm, Sweden
Relapse variants in chronic Plasmodium falciparum infections are antigenically
distinct from the parental parasites. The variable antigen PfEMP1 expressed at
the surface of the infected erythrocyte (IE) is encoded by the var gene family
with approximately 60 copies per haploid genome. Placental isolates commonly
express DBLgamma containing subtypes of var genes with homology to either
3D7var5.2 (var(COMMON)) or FCR3var(CSA). Here we report that var(COMMON)
related genes are constitutively transcribed in approximately 60% of malaria infected
children in Gabon. var(COMMON) is conserved in field isolates over at least
2.1kb. In 3D7 parasites var(COMMON) is present on chromosome 5 (var5.2) and
constitutively transcribed in the opposite direction to most other var genes. It
lacks a regulatory intron, an acidic terminal segment and ends in telomeric
repeat sequences. var(COMMON) encodes a large, hypothetical PfEMP1 of a
structure similar to previous placenta-binding PfEMP1s but it is not present at
the IE-surface. IE of a 3D7 clone (3D7S8) transcribe var(COMMON) but express a
PfEMP1 distinct from var(COMMON) at the surface and adhere to placental tissues
through var(COMMON) independent novel mechanisms. Our report suggests that
expression of var(COMMON) type genes is not restricted to placental malaria.
Parasite 2003 Mar;10(1):39-50
The treatment of Plasmodium falciparum-infected erythrocytes with
chloroquine leads to accumulation of ferriprotoporphyrin IX bound to
particular parasite proteins and to the inhibition of the parasite's
Famin O, Ginsburg H.
Department of Biological Chemistry, Institute of Life Sciences, Hebrew
University of Jerusalem, Jerusalem 91904, Israel.
Ferriprotoporphyrin IX (FPIX) is a potentially toxic product of hemoglobin
digestion by intra-erythrocytic malaria parasites. It is detoxified by
biomineralization or through degradation by glutathione. Both processes are
inhibited by the antimalarial drug chloroquine, leading to the accumulation of
FPIX in the membranes of the infected cell and their consequent
permeabilization. It is shown here that treatment of Plasmodium
falciparum-infected erythrocytes with chloroquine also leads to the binding of
FPIX to a subset of parasite proteins. Parasite enzymes such as aldolase,
pyrimidine nucleaside monophosphate kinase and pyrimidine 5'-nucleotidase were
inhibited by FPIX in vitro, but only the activity of 6-phosphogluconate
dehydrogenase was reduced significantly in cells after drug treatment.
Additional proteins were extracted from parasite cytosol by their ability to
bind FPIX. Sequencing of these proteins identified heat shock proteins 90 and
70, enolase, elongation factor 1-alpha, phoshoglycerate kinase, glyceraldehyde
3-phosphate dehydrogenase, L-lactate dehydrogenase and gametocytogenesis
onset-specific protein. The possible involvement of these proteins in the
antimalarial mode of action of chloroquine is discussed. It is concluded that
drug-induced binding of FPIX to parasite glycolytic enzymes could underlie the
demonstrable inhibition of glycolysis by chloroquine. The inhibition of
6-phosphogluconate dehydrogenase could explain the reduction of the activity of
the hexose monophosphate shunt by the drug. Inhibition of both processes is
deleterious to parasite survival. Binding of FPIX to other proteins is probably
inconsequential to the rapid killing of the parasite by chloroquine.
J Biol Chem 2003 Mar 31;
Multiple splice variants encode a novel adenylyl cyclase of possible plastid
origin expressed in the sexual stage of the malaria parasite plasmodium
Muhia DK, Swales CA, Eckstein-Ludwig U, Saran S, Polley SD, Kelly JM, Schaap P,
Krishna S, Baker DA.
Department of Infectious and Tropical Diseases, London School of Hygiene and
Tropical Medicine, London WC1E 7HT.
We report the characterisation of an unusual adenylyl cyclase gene from
Plasmodium falciparum, here designated PfACalpha. The level of mRNA expression
is maximal during development of gametocytes (the sexual blood stage of the
parasite life cycle). The gene is highly interrupted by 22 introns, and RT-PCR
analysis revealed that there are multiple mRNA splice variants. One intron has
three alternative 3 splice sites which confer the potential to encode distinct
forms of the enzyme using alternative start codons. Deduced amino acid sequences
predict membrane-spanning regions, the number of which can vary between two and
six depending on the splice variant. Expression of a synthetic form of two of
these variants in Xenopus oocytes and in Dictyostelium adenylyl
cyclase-deficient mutants, confirms that PfACalpha is a functional adenylyl
cyclase. These results identify a novel mechanism in P. falciparum for the
generation of multiple isoforms of a key, membrane-bound signalling molecule
from a single genomic copy. Comparisons of the catalytic domains of PfACalpha
and a second putative P. falciparum adenylyl cyclase (PfACbeta) with those from
other species reveal an unexpected similarity with adenylyl cyclases from
certain prokaryotes including the cyanobacteria (blue green algae). In addition,
the presence of an unusual active site substitution in a position that
determines substrate specificity, also characteristic of these prokaryotic
forms, further suggests a plastid origin for the Plasmodium enzymes.
Trop Med Int Health 2003 Apr;8(4):310-315
The effect on haemoglobin of the use of iron cooking pots in rural Malawian
households in an area with high malaria prevalence: a randomized trial.
Geerligs PP, Brabin B, Mkumbwa A, Broadhead R, Cuevas LE.
Tropical Child Health Group, Division of Tropical Medicine, Liverpool School of
Tropical Medicine, Liverpool, UK Department of Paediatrics, Medical College,
Blantyre, Malawi Emma Kinderziekenhuis, Academic Medical Centre, University of
Amsterdam, The Netherlands.
BACKGROUND: Innovative low-cost sustainable strategies are required to reduce
the high prevalence of iron-deficiency anaemia in developing countries. METHODS:
We undertook a community-based randomized controlled intervention trial to
assess the effects of cooking in iron or aluminium cooking pots in Malawian
households in an area with high malaria prevalence. Analysis was by intention to
treat and consistency of use. The primary outcomes were change in haemoglobin
and iron status. FINDINGS: The study population comprised 164 participants
eating from aluminium cooking pots and 158 from iron cooking pots. The mean
haemoglobin change was significantly increased after 6 weeks in adults who
consistently ate from an iron cooking pot (+3.6 g/l compared to -3.2 g/l, mean
difference between groups 6.8 g/l, 95% CI +0.86, +12.74). In children, no
significant haemoglobin change was observed in consistent pot users, although
they showed a significant reduction in iron deficiency (iron 8.6 ZP/g and
aluminium 10.8 ZP/g, mean difference 2.2 ZP/g, 95% CI +1.08, +3.32).
INTERPRETATION: Rural Malawian adults in a high malaria transmission area who
consistently consume food prepared in iron cooking pots show a significant rise
in haemoglobin after 6 weeks use. Children showed a reduction in iron
deficiency, but no significant improvement in haemoglobin, possibly because of
their high malaria parasite prevalence. Using iron cooking pots in developing
countries could provide an innovative way to prevent iron deficiency and anaemia
in malarious areas where regular iron supplementation is problematic.
Trop Med Int Health 2003 Apr;8(4):297-300
Malaria in Mauritania: the first cases of malaria endemic to Nouakchott.
Cortes H, Morillas-Marquez F, Valero A.
Department of Parasitology, Faculty of Pharmacy, University of Granada, Spain
Laboratory of the Polyclinic of Nouakchott, Nouakchott, Mauritania.
The current situation of endemic malaria in Mauritania is not clear since, in
most health centres, suspected malaria cases are not confirmed by
parasitological analysis and diagnosis is based on clinical symptoms alone. To
obtain reliable data about malaria in this country, thin and thick blood smears
were taken from patients with symptoms compatible with the illness, who attended
two hospitals: Polyclinic of Nouakchott, which serves one-third of the country's
population, where a malaria infection rate of 18.5% (77 of 446) was recorded;
Plasmodium falciparum caused 61.85% of these, P. vivax 35.5% (28/77). In Kaedi
Regional Hospital, provincial capital of the endemic Gorgol region, a prevalence
of 25.49% (106 of 416) was recorded, with P. falciparum as the sole pathogenic
species. Of the 77 cases of malaria diagnosed in Nouakchott, nine (seven of P.
falciparum and two of P. vivax) were considered as endemic to the city. These
cases were all children under 8 years of age except for one adult who had never
left the capital, and this is the first time that cases endemic to this city
have been detected. PMID: 12667147
West Afr J Med 2002 Oct-Dec;21(4):286-7
Therapeutic monitoring of chloroquine in pregnant women with malaria.
Fakeye TO, Fehintola FA, Ademowo OG, Walker O.
Department of Pharmaceutical Microbiology & Clinical Pharmacy, College of
Medicine, University of Ibadan, Nigeria.
Therapeutic monitoring of chloroquine was carried out in pregnant women with
confirmed laboratory and clinical malaria after administration of 10 mg/kg body
weight of the drug at 0 and 24th hour and 5 mg/kg body weight at the 48th hour.
Venous blood was withdrawn at scheduled intervals. Plasma chloroquine level was
determined using a highly sensitive and specific liquid chromatographic method.
The time of peak plasma concentration, (t(max)) after the first dose was found
to be 3.5 hours while peak plasma concentrations, (Cp(max)) were obtained at 2,
28, and 52 hours with values of 204. 36, 343.51 and 257.04 ng/ml respectively.
There was total parasitaemia clearance before the end of 96 hours in all the
subjects. PMID: 12665265
West Afr J Med 2002 Oct-Dec;21(4):276-9
Low plasma bicarbonate predicts poor outcome of cerebral malaria in
Oguche S, Omokhodion SI, Adeyemo AA, Olumese PE.
Department of Paediatrics, College of Medical Sciences, University of Maiduguri,
Malaria remains a major cause of morbidity and mortality in many sub Saharan
countries and cerebral malaria is widely recognised as one of its most fatal
forms. We studied the predictive value of routine biochemical laboratory indices
in predicting the outcome of cerebral malaria in 50 Nigerian children ages 9
months to 6 years with cerebral malaria at the University College Hospital,
Ibadan, Nigeria. Of the 50 children studied, 43 (68%) made a full recovery, 5
(105) developed neurological sequelae while 11(22%) died. Biochemical
derangements observed among the children included azotaemia (29%), elevated
plasma creatinine (20%), metabolic acidiosis (22%) and hyponatraemia (16%).
Metabolic acidosis and elevated plasma creatinine on admission were
significantly associated with a poor outcome (p < 0.05). Hyponatraemia and
hypokalaemia were not significantly associated with outcome. On multivariated
analysis, metabolic acidosis and elevated plasma creatinine on admission to
hospital remained independent predictors of poor outcome after adjusting for
other known risk factors. Patients with these findings require prompt referral
for adequate treatment in centres equipped to manage such critically ill
patients. PMID: 12665262
J Forensic Sci 2003 Mar;48(2):404-8
Malaria deaths in the United States: case report and review of deaths,
Stoppacher R, Adams SP.
Milwaukee County Medical Examiner's Office, Milwaukee, WI 53233, USA.
Malaria is the world's most important parasitic disease, accounting for an
estimated 300 to 500 million new cases and between 1.5 and 2.7 deaths annually.
The majority of these deaths occur in sub-Saharan Africa where malaria is
endemic and are the result of infection with Plasmodium falciparum. The number
of deaths in the United States due to malaria is comparably much lower and
involves so-called "imported" cases in which U.S. travelers acquire the
infection upon travel to endemic areas and subsequently return to the United
States or in which infected foreign citizens travel to the United States. There
were a total of 118 deaths due to malaria in the United States between 1979 and
1998 with an average of 5.9 deaths per year. Specific epidemiological data
provided by the CDC regarding the 40 deaths that occurred between 1992 and 1998
yielded the following results. Deaths occurred in patients ranging from 9 months
to 89 years of age (median, 53 years). Thirty-eight (95%) of these were due to
P. falciparum and two (5%) due to P. vivax. Anti-malarial chemoprophylaxis was
taken in 40% of cases, not taken in 45% of cases, and unknown in 15% of cases.
Twenty-four (60%) of the cases involved U.S. travelers to endemic areas, of whom
59% traveled to Africa, 25% to South America, 8% to India, 4% to Haiti, and 4%
to unspecified areas. The remaining cases included eleven foreign travelers to
the U.S. (27.5%), three induced cases (7.5%), and two undetermined cases (5%).
Thirty-nine (98%) of the cases were diagnosed antemortem and only one case was
known to have come to the attention of the medical examiner/coroner. An
illustrative case report demonstrates many of the features associated with fatal
malaria infections in the United States. The case involves a U.S. student who
was studying in Africa and who, by report, had not taken antimalarial
chemoprophylaxis. Despite seeking medical attention, the patient was not
diagnosed with P. falciparum infection and cerebral malaria until the time of
medico-legal autopsy, where the classic gross and microscopic features of
cerebral malaria were identified. This case represents one of the few cases of
P. falciparum infection in the United States not diagnosed antemortem. Given the
worldwide prevalence of the disease, increasing international travel, and
rapidly developing drug resistance, malaria will continue to be an important
disease and should be considered in cases of sudden, unexplained deaths. By
reviewing the major epidemiological features of malaria-related deaths in the
United States and by presenting the major gross and microscopic features of
cerebral malaria, an attempt is made at raising the awareness of the forensic
community to the potential of malaria-related deaths. PMID: 12665001
Ann Trop Med Parasitol 2003 Jan;97(1):15-21
A single-step, PCR-based method for the detection and differentiation of
Plasmodium vivax and P. falciparum.
Patsoula E, Spanakos G, Sofianatou D, Parara M, Vakalis NC.
Department of Parasitology, Entomology and Tropical Diseases, National School of
Public Health, 196 Alexandras Avenue, 11521, Athens, Greece.
Email: [email protected]
The ability to detect and differentiate between Plasmodium falciparum and P.
vivax is of great importance for the routine laboratory diagnosis of malaria,
donor-blood screening and epidemiological studies. Most PCR-based methods for
the discrimination of these two species require nested protocols or an
additional hybridization reaction, leading to high labour costs and long
turn-around times. A simple, time-effective and yet sensitive and specific
technique, based on a multiplex PCR, has now been developed for the simultaneous
detection and differentiation of P. falciparum and P. vivax in blood samples.
Compared with the 'gold standard' of microscopy, this method had a sensitivity
and specificity of 100%, with a detection limit of just one P. falciparum or
three P. vivax parasites/microl blood. PMID: 12662418
Ann Trop Med Parasitol 2003 Jan;97(1):5-13
The hospital- and field-based performances of the OptiMAL test, for malaria
diagnosis and treatment monitoring in central India.
Singh N, Valecha N, Nagpal AC, Mishra SS, Varma HS, Subbarao SK.
Malaria Research Centre (Field Station), NSCB Medical College Building, Jabalpur
- 482003, India.
The performance of the OptiMAL test, to detect and differentiate Plasmodium
falciparum and P. vivax, was evaluated in central India. The subjects were
either symptomatic patients, who presented at a referral hospital in urban
Jabalpur, or the inhabitants of remote, tribal, forested villages where malaria
is a major public-health problem. In each setting, the results of conventional
microscopy were used as the 'gold standard'. Under hospital conditions, the test
had excellent sensitivity (100%), good specificity (97%), a high positive
predictive value (98%) and a high negative predictive value (100%). The
corresponding values in the field-based study in the tribal villages (100%, 67%,
84% and 100%, respectively) were almost as good. The results of OptiMAL testing
reveal the decline in parasitaemias (of P. falciparum or P. vivax) after drug
administration. For monitoring the effectiveness of treatment, the test could
therefore be a useful alternative to microscopy, particularly (1) in places
where the facilities for microscopy are poor or non-existent and (2) among
hospitalized patients with severe, complicated malaria (in whom parasitaemia and
drug response need to be followed very carefully). Follow-up (within 28 days of
diagnosis) of the 58 malaria cases detected in the field revealed that the
OptiMAL test can be used to detect re-infection with a different Plasmodium sp.
(sensitivity=100%; specificity=100%; J-index=1) or recrudescence/re-infection
with the same Plasmodium sp. (sensitivity=83%; specificity=100%; J-index=0.83)
accurately. The ability to use the test to distinguish P. falciparum from P.
vivax, and to identify mixed infections of these two species, is of great
significance in areas where the preferred and effective therapy for P.
falciparum malaria differs from that for P. vivax. PMID: 12662417
J Infect Dis 2003 Apr 1;187(7):1137-41
Antibody Responses to Plasmodium falciparum Merozoite Surface Protein-1
and Efficacy of Amodiaquine in Gabonese Children with P. falciparum Malaria.
Mawili-Mboumba DP, Borrmann S, Cavanagh DR, McBride JS, Matsiegui PB, Missinou
MA, Kremsner PG, Ntoumi F.
Unite de Recherche Medicale, Hopital Albert Schweitzer, Lambarene, Gabon, and
Sektion Humanparasitologie, Institut fur Tropenmedizin, Universitat Tubingen,
The relationship between the efficacy of amodiaquine for the treatment of
uncomplicated Plasmodium falciparum malaria and preexisting antibodies against
merozoite surface protein (MSP)-1, a blood-stage P. falciparum antigen, was
investigated. The immunoglobulin G antibody response to different MSP-1
recombinant proteins was evaluated in plasma samples from Gabonese children with
uncomplicated malaria who were treated with amodiaquine. The prevalence of
anti-MSP-1 antibodies was similar among patients with either parasitological and
clinical cure after treatment (n=102) or treatment failure (n=51) by day 28 (83%
in both groups). However, associations between antibody responses to K1 and
MAD20 allelic families and therapeutic success were found (P<.001 and P=.034,
respectively). A high proportion of plasma samples recognizing several antigens
was found in the cured group. This association was significant even when data
were stratified by age, particularly for the K1 family antigens (P=.029). These
results suggest that humoral immune responses play a supportive role in the
efficacy of amodiaquine treatment. PMID: 12660928
Acta Trop 2003 Mar;85(3):363-73
High prevalence of quintuple mutant dhps/dhfr genes in Plasmodium
falciparum infections seven years after introduction of sulfadoxine and
pyrimethamine as first line treatment in Malawi.
Bwijo B, Kaneko A, Takechi M, Zungu IL, Moriyama Y, Lum JK, Tsukahara T, Mita T,
Takahashi N, Bergqvist Y, Bjorkman A, Kobayakawa T.
Department of International Affairs and Tropical Medicine, Tokyo Women's Medical
University, 8-1 Kawada-Cho, Shinjuku-Ku, 162 8666, Tokyo, Japan
Malawi changed its national policy for malaria treatment in 1993, becoming the
first country in Africa to replace chloroquine by sulfadoxine and pyrimethamine
combination (SP) as the first-line drug for uncomplicated malaria. Seven years
after this change, we investigated the prevalence of dihydropteroate synthase
(dhps) and dihydrofolate reductase (dhfr) mutations, known to be associated with
decreased sensitivity to SP, in 173 asymptomatic Plasmodium falciparum
infections from Salima, Malawi. A high prevalence rate (78%) of parasites with
triple Asn-108/Ile-51/Arg-59 dhfr and double Gly-437/Glu-540 dhps mutations was
found. This 'quintuple mutant' is considered as a molecular marker for clinical
failure of SP treatment of P. falciparum malaria. A total of 11 different dhfr
and dhps combinations were detected, 3 of which were not previously reported.
Nineteen isolates contained the single Glu-540 mutant dhps, while no isolate
contained the single Gly-437 mutant dhps, an unexpected finding since Gly-437
are mostly assumed to be one of the first mutations commonly selected under
sulfadoxine pressure. Two isolates contained the dhps single or double mutant
coupled with dhfr wild-type. The high prevalence rates of the three dhfr
mutations in our study were consistent with a previous survey in 1995 in
Karonga, Malawi, whereas the prevalences of dhps mutations had increased, most
probably as a result of the wide use of SP. A total of 52 P. falciparum isolates
were also investigated for pyrimethamine and sulfadoxine/pyrimethamine activity
against parasite growth according to WHO in vitro standard protocol. A
pyrimethamine resistant profile was found. When pyrimethamine was combined with
sulfadoxine, the mean EC(50) value decreased to less than one tenth of the
pyrimethamine alone level. This synergistic activity may be explained by
sulfadoxine inhibition of dhps despite the double mutations in the dhps genes,
which would interact with pyrimethamine acting to block the remaining folate
despite dhfr mutations in the low p-aminobenzoic acid and low folic acid medium
mixed with blood. PMID: 12659974
Acta Trop 2003 Mar;85(3):355-361
Relationship between entomological inoculation rate, Plasmodium falciparum
prevalence rate, and incidence of malaria attack in rural Gabon.
Elissa N, Migot-Nabias F, Luty A, Renaut A, Toure F, Vaillant M, Lawoko M,
Yangari P, Mayombo J, Lekoulou F, Tshipamba P, Moukagni R, Millet P, Deloron P.
International Center for Medical Research, BP 769, Franceville, 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/&mgr;l 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. PMID: 12659973
J Parasitol 2003 Feb;89(1):190-2
Manipulation of the vertebrate host's testosterone does not affect gametocyte
sex ratio of a malaria parasite.
Osgood SM, Eisen RJ, Wargo AR, Schall JJ.
Department of Biology, University of Vermont, Burlington, Vermont 05405, USA.
Gametocyte sex ratio of the malaria parasite Plasmodium mexicanum is variable in
its host, the western fence lizard (Sceloporus occidentalis), both among
infections and within infections over time. We sought to determine the effect of
host physiological quality on the gametocyte sex ratio in experimentally induced
infections of P. mexicanum. Adult male lizards were assigned to 4 treatment
groups: castrated, castrated + testosterone implant, sham implant, and
unmanipulated control. No significant difference in gametocyte sex ratio was
found among the 4 treatment groups. Two other analyses were performed. A surgery
stress analysis compared infection sex ratio of castrated, castrated +
testosterone implant, and sham implant groups with the unmanipulated control
group. A testosterone alteration analysis compared infection sex ratio of the
castrated and castrated + testosterone implant groups with the sham implant and
unmanipulated control groups. Again, no significant difference was observed for
these 2 comparisons. Thus, physiological changes expected for experimentally
induced variation in host testosterone and the stress of surgery were not
associated with any change in the gametocyte sex ratio. Also, theex-periment
suggests testosterone is not a cue for shaping the sex ratio of gametocytes in
P. mexicanum. These results are related to the evolutionary theory of sex ratios
as applied to malaria parasites. PMID: 12659329
Wilderness Environ Med 2003 Spring;14(1):17-9
Accurate clinical diagnosis of malaria in a postflood epidemic: a field
study in Mozambique.
Loveridge BW, Henner JR, Lee FC.
Department of Emergency Medicine, Wright State University School of Medicine,
Dayton, OH, USA.
OBJECTIVE: To determine the positive predictive value (PPV) of a clinical
diagnosis vs laboratory testing for malaria in a remote field setting following
a natural disaster. METHODS: During an observational study, as part of a
disaster response in Mozambique, patients were clinically diagnosed and treated
for malaria. The population included native tribes in remote areas and displaced
persons in refugee camps representing all age groups, male and female. The
diagnosis was made if patients exhibited at least 3 of the following historical
features: fever, chills, headache, and nausea/vomiting (N/V). In addition they
had to have either a tactile fever or palpable spleen. At the conclusion of the
mission, 28 patients were prospectively tested with an antigen-capture assay for
P. falciparum to determine the PPV of clinical diagnosis vs definitive
laboratory testing. RESULTS: During the study period, 1215 of 4064 (30%)
patients were diagnosed with malaria based on clinical presentation. On our
final day, 28 consecutive patients with a clinical diagnosis of malaria were
tested using an antigen-capture assay for P. falciparum. Of those, 25 tested
positive-yielding a PPV of 89% (CI 0.78-1.01). CONCLUSION: In a remote field
setting where malaria is endemic/epidemic, diagnosis of malaria based on
selected historical and physical findings is possible with a high positive
predictive value. PMID: 12659244
Wien Klin Wochenschr 2003 Jan 31;115(1-2):63-5
Severe malaria in a splenectomised Gabonese woman.
Grobusch MP, Borrmann S, Omva J, Issifou S, Kremsner PG.
Medical Research Unit, Albert Schweitzer Hospital, Lambarene, Gabon.
Email: [email protected]
BACKGROUND: The intact splenic function is of utmost importance for the host's
defence capacity against Plasmodium spp. not only by limiting the acute
infection through the removal of parasites from the blood stream, but also by
modulating parasite antigen expression on the surface of infected red blood
cells as well as cellular and humoral immune response. Splenectomised
individuals are at high risk to develop a more severe and prolonged disease,
even if they had acquired semi-immunity prior to their loss of splenic tissue.
CASE REPORT: We report on a 37 year old splenectomised Gabonese woman who
developed severe falciparum malaria with hyperparasitaemia, profound anaemia,
and an overwhelming gametocytaemia, recovering very slowly following quinine
therapy. Whereas the clinical course is not at odds with previous descriptions,
the massive occurrence of mature gametocytes as documented here has not been
reported before. Whether the high gametocyte count observed in our patient was
primarily due to the impaired clearance of asexual forms or to the induction of
gametocytogenesis remains unclear. Regarding the optimal drug regimen for
treating malaria in splenectomized patients, a combination of an aminoquinoline
with an artemisinin derivative might be the optimal choice. PMID: 12658914
Mol Genet Genomics 2003 Mar;268(6):720-8
Construction of a BAC library and generation of BAC end sequence-tagged
connectors for genome sequencing of the African malaria mosquito
Hong YS, Hogan JR, Wang X, Sarkar A, Sim C, Loftus BJ, Ren C, Huff ER, Carlile
JL, Black K, Zhang HB, Gardner MJ, Collins FH.
Center for Tropical Disease Research and Training, Department of Biological
Sciences, University of Notre Dame, IN 46556, Notre Dame, USA.
A Bacterial Artificial Chromosome (BAC) genomic DNA library of Anopheles
gambiae, the major human malaria vector in sub-Saharan Africa, was constructed
and characterized. This library (ND-TAM) is composed of 30,720 BAC clones in
eighty 384-well plates. The estimated average insert size of the library is 133
kb, with an overall genome coverage of approximately 14-fold. The ends of
approximately two-thirds of the clones in the library were sequenced, yielding
32,340 pair-mate ends. A statistical analysis (G-test) of the results of PCR
screening of the library indicated a random distribution of BACs in the genome,
although one gap encompassing the whitelocus on the X-chromosome was identified.
Furthermore, combined with another previously constructed BAC library (ND-1),
~2,000 BACs have been physically mapped by polytene chromosomal in situ
hybridization. These BAC end pair mates and physically mapped BACs have been
useful for both the assembly of a fully sequenced A. gambiaegenome and for
linking the assembled sequence to the three polytene chromosomes. This ND-TAM
library is now publicly available at both http://www.malaria.mr4.org/mr4pages/index.html/
and http://hbz.tamu.edu/, providing a valuable resource to the mosquito research community.
Infect Immun 2003 Apr;71(4):2032-40
Induction of Mosquitocidal Activity in Mice Immunized with Anopheles
gambiae Midgut cDNA.
Foy BD, Magalhaes T, Injera WE, Sutherland I, Devenport M, Thanawastien A,
Ripley D, Cardenas-Freytag L, Beier JC.
Department of Tropical Medicine. Department of Microbiology and Immunology,
Tulane University, New Orleans, Louisiana. Department of Genetics, Case Western
Reserve University, Cleveland, Ohio.
Vaccines that induce mosquito-killing (mosquitocidal) activity could
substantially reduce the transmission of certain mosquito-borne diseases,
especially vaccines against African malaria vectors, such as the mosquito
Anopheles gambiae. To generate and characterize antimosquito immunity we
immunized groups of mice with two individual A. gambiae midgut cDNAs, Ag-Aper1
(a secreted peritrophic matrix protein) and AgMuc1 (a midgut-bound mucin), and
an A. gambiae midgut cDNA library from blood-fed mosquitoes. We observed
significantly increased mortality among mosquitoes that fed on either the
AgMuc1- or the cDNA library-immunized mice compared to that of controls, but no
differences were observed among those fed on Ag-Aper1-immunized mice. Analysis
of the humoral and cellular immune responses from mice showed that the induced
mosquitocidal effect was associated with immune profiles characterized by
elevated tumor necrosis factor alpha and gamma interferon cytokine levels and
very low antibody titers. Furthermore, an additional immunization of cDNA
library-immunized mice with midgut protein shifted immunity toward a Th2-type
immune response, characterized by elevated antibody titers and high
interleukin-5 and interleukin-10 cytokine levels; importantly, mosquitoes
feeding on these mice exhibited no undo mortality. Finally, when immune sera was
ingested by mosquitoes through a membrane feeder, no effect on mosquito
mortality was observed, indicating that serum factors alone were not responsible
for the mosquitocidal effect. Our results demonstrate that mosquitocidal
immunity in mice can be consistently generated by midgut cDNA immunization and
suggest this cDNA-induced mosquitocidal immunity is cell mediated.
Infect Immun 2003 Apr;71(4):1911-8
P-selectin contributes to severe experimental malaria but is not required for
leukocyte adhesion to brain microvasculature.
Chang WL, Li J, Sun G, Chen HL, Specian RD, Berney SM, Granger DN, van der Heyde
Department of Medicine, Louisiana State University Health Sciences Center,
Shreveport, LA 71130, USA.
Plasmodium berghei-infected mice, a well-recognized model of experimental
cerebral malaria (ECM), exhibit many of the hallmarks of a systemic inflammatory
response, with organ damage in brain, lung, and kidneys. Identification of the
molecules mediating pathogenesis of the inflammatory response, such as leukocyte
adhesion, may lead to new therapies. Indeed, mice lacking the cell adhesion
molecule P-selectin were significantly (P = 0.005) protected from death due to
P. berghei malaria compared with C57BL/6 controls despite similar parasitemia (P
= 0.6) being found in both groups of mice. P-selectin levels assessed by the
quantitative dual radiolabeled monoclonal antibody technique increased
significantly (P < 0.05) in several organs in C57BL/6 mice infected with P.
berghei, supporting the concept of a systemic inflammatory response mediating
malarial pathogenesis. Intravital microscopic analysis of the brain
microvasculature demonstrated significant (P < 0.001) leukocyte rolling and
adhesion in brain venules of P. berghei-infected mice compared with those found
in uninfected controls. The maximum leukocyte adhesion occurred on day 6 of P.
berghei infection, when the mice become moribund and exhibit marked vascular
leakage into the brain, lung, and heart. However, P-selectin levels were
significantly (P < 0.005) increased in brain, lung, and kidneys during P.
berghei malaria in ECM-resistant BALB/c mice compared with those found in
uninfected BALB/c controls, indicating that increased P-selectin alone is not
sufficient to mediate malarial pathogenesis. Leukocyte adhesion to brain
microvessels of P-selectin-deficient mice with P. berghei malaria was similar to
that observed in control mice. Collectively, these results indicate that
P-selectin is important for the development of malarial pathogenesis but is not
required for leukocyte adhesion in brain. PMID: 12654808
Infect Immun 2003 Apr;71(4):1833-42
Repeat sequences in block 2 of Plasmodium falciparum merozoite surface
protein 1 are targets of antibodies associated with protection from malaria.
Polley SD, Tetteh KK, Cavanagh DR, Pearce RJ, Lloyd JM, Bojang KA, Okenu DM,
Greenwood BM, McBride JS, Conway DJ.
London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT,
Human antibodies to the block 2 region of Plasmodium falciparum merozoite
surface protein 1 (MSP1) are associated with a reduced prospective risk of
clinical malaria. Block 2 is highly polymorphic, but all known alleles can be
grouped into three major types. Two of these types (the K1-like and MAD20-like
types) contain type-specific sequences (found in all alleles of a particular
type) that flank polymorphic tripeptide repeats. These repeats contain both
type-specific and subtype-specific sequences. To evaluate the antibody
recognition of these parts of block 2, a new panel of six recombinant proteins
was used (fused type-specific flanking sequences and two representative repeat
sequences for each of the K1-like and MAD20-like types separately). Extensive
testing of these antigens and full-length block 2 antigens showed that human
serum immunoglobulin G antibodies induced by infection can recognize (i)
type-specific epitopes in the repeats, (ii) subtype-specific epitopes in the
repeats, or (iii) type-specific epitopes in flanking sequences. A large
prospective study in The Gambia showed that antibodies to the repeats are
strongly associated with protection from clinical malaria. The results are
important for design of a vaccine to induce protective antibodies, and they
address hypotheses about repeat sequences in malaria antigens.
J Antimicrob Chemother 2003 Apr;51(4):1021-4
In vitro susceptibility to a new antimalarial organometallic analogue,
ferroquine, of Plasmodium falciparum isolates from the Haut-Ogooue
region of Gabon.
Atteke C, Ndong JM, Aubouy A, Maciejewski L, Brocard J, Lebibi J, Deloron P.
Universite des Sciences et Techniques de Masuku (USTM), Franceville.
OBJECTIVES: To assess the activity of a new organometallic chloroquine analogue,
ferroquine, against numerous Plasmodium falciparum isolates from Gabon.
METHODS: The in vitro susceptibility of 116 P. falciparum isolates to chloroquine and
ferroquine was assessed using the isotopic microtest. All isolates were from
outpatients in the Franceville and Bakoumba medical centres in the province of
Haut-Ogooue, south-east Gabon. RESULTS: The in vitro resistance to chloroquine
was 51.8% in Franceville and 96.7% in Bakoumba. The IC50 geometric mean
(95% CI) of ferroquine against isolates in Franceville was 16.0 (14.4-17.8) nM, with
individual values ranging from 1.0 to 47.0 nM; in Bakoumba it was 27.9
(23.4-33.2) nM, with individual values ranging from 1.0 to 62.0 nM. Compared
with chloroquine, ferroquine was 5.3 times more active on isolates susceptible
to chloroquine, and 13.3 times more active on isolates resistant to chloroquine.
A weak positive correlation was observed between responses of these two drugs,
but too low to demonstrate cross-resistance. CONCLUSIONS: Ferroquine may be
useful as an alternative drug for treating chloroquine-resistant malaria.
Antimicrob Agents Chemother 2003 Apr;47(4):1347-54
Molecular Determination of Point Mutation Haplotypes in the
Dihydrofolate Reductase and Dihydropteroate Synthase of Plasmodium
falciparum in Three Districts of Northern Tanzania.
Pearce RJ, Drakeley C, Chandramohan D, Mosha F, Roper C.
Department of Infectious and Tropical Diseases, London School of Hygiene and
Tropical Medicine, London, WC1E 7HT, United Kingdom. Kilimanjaro Christian
Medical Centre and. The Joint Malaria Programme (JMP) Moshi, Tanzania.
The antimalarial combination of sulfadoxine and pyrimethamine (SP) was
introduced as first-line treatment for uncomplicated malaria in Tanzania during
2001 following 18 years of second-line use. The genetic determinants of in vitro
resistance to the two drugs individually are shown to be point mutations at
seven sites in the dihydrofolate reductase gene (dhfr) conferring resistance to
pyrimethamine and five sites in the dihydropteroate synthase (dhps) gene
conferring resistance to sulfadoxine. Different combinations of mutations within
each gene confer differing degrees of insensitivity, but information about the
frequency with which allelic haplotypes occur has been lacking because of the
complicating effects of multiple infection. Here we used a novel high-throughput
sequence-specific oligonucleotide probe-based approach to examine the present
resistance status of three Plasmodium falciparum populations in northern
Tanzania. By using surveys of asymptomatic infections and screening for the
presence of all known point mutations in dhfr and dhps genes, we showed that
just five dhfr and three dhps allelic haplotypes are present. High frequencies
of both triple-mutant dhfr and double-mutant dhps mutant alleles were found in
addition to significant interregional heterogeneity in allele frequency. In vivo
studies have shown that the cooccurrence of three dhfr mutations and two dhps
mutations in an infection prior to treatment is statistically predictive of
treatment failure. We have combined data for both loci to determine the
frequency of two-locus genotypes. The triple-dhfr/double-dhps genotype is
present in all three regions with frequencies ranging between 30 and 63%,
indicating that treatment failure rates are likely to be high.
Vector Borne Zoonotic Dis 2001 Fall;1(3):231-7
Estimation of vectorial capacity of Anopheles dirus (Diptera: Culicidae) in a
forest-fringed village of Assam (India).
Prakash A, Bhattacharyya DR, Mohapatra PK, Mahanta J.
Regional Medical Research Centre, NE Region (Indian Council of Medical
Research), Dibrugarh, Assam, India.
To estimate the vectorial capacity of Anopheles dirus, the main vector of forest
malaria in the northeastern region of India, in order to gain an understanding
of entomological factors related to malaria transmission in forest-fringe areas
of Assam, India, an isolated village in the tropical rain forest-fringed area in
the district of Dibrugarh, Assam, under the influence of An. dirus alone was
studied. Data on various entomological variables required for computation of the
vectorial capacity were generated in each month from June 1999 to May 2000 in
the field using standard techniques. Malaria prevalence was also studied during
the same period in the study village and correlated with the estimated vectorial
capacity of An. dirus. Vectorial capacity of An. dirus was highest, 0.779 for
Plasmodium vivax (Pv) and 0.649 for Plasmodium falciparum (Pf), during the
hot-monsoon season (June-September) and decreased to 0.08 (Pv) and 0.07(Pf) in
the temperate postmonsoon season (October-November) before attaining zero values
in the cool-dry season (December-February). With increasing temperature in the
temperate premonsoon season (March-May), vectorial capacity recorded was 0.119
and 0.82 for Pv and Pf, respectively. Significant positive correlation was seen
between the estimated vectorial capacity of An. dirus and the number of new Pf
(r = 0.86, p < 0.001) and Pv (r = 0.69, p < 0.02) cases in the study village in
different months. Thus, this study highlights the pattern of malaria
transmission by An. dirus in a forest-fringe area of Assam that begins in March,
peaks in July/August, subsides by November, and remains interrupted between
December and February. Measures for controlling malaria in forest-fringe areas
should be scheduled accordingly. PMID: 12653151
J Chromatogr B Analyt Technol Biomed Life Sci 2003 Mar 25;786(1-2):61-72
Expression and purification of Plasmodium falciparum MSP-1(42): A malaria
Epp C, Kauth CW, Bujard H, Lutz R.
Zentrum fur Molekulare Biologie der Universitat Heidelberg (ZMBH), Im
Neuenheimer Feld 282, D-69120, Heidelberg, Germany
The C-terminal 42.10(3) Da portion of the merozoite surface protein (MSP-1) of
the human malaria parasite Plasmodium falciparum is of interest, not only
because it may constitute an essential part of a future anti-malaria vaccine,
but also due to its role during the infection of erythrocytes by the parasite.
We have cloned and expressed two synthetic DNA sequences encoding the two
prototypic MSP-1(42) variants in E. coli. When over-produced, both proteins form
insoluble aggregates which were isolated in high purity and yield. After
solubilisation and refolding in vitro, both proteins were purified to
homogeneity by a three-step procedure applying Ni-chelate, size exclusion and
immuno-affinity chromatography. After purification, both proteins meet key
criteria of preparations for clinical use. First, conformational studies suggest
proper folding of the proteins, particularly in the region containing two
EGF-like domains. Polyclonal serum raised against E. coli produced MSP-1(42)
recognizes native MSP-1 in Plasmodium infected erythrocytes as shown by
immunofluorescence. PMID: 12651002
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