Molecular epidemiology of Plasmodium vivax in the State of Amazonas, Brazil.

Over the past 2 decades, the Amazon region of Brazil has experienced reemergence of Plasmodium vivax malaria, with reported occurrence of severe disease. The frequency and manifestations of this severe disease are unlike previous clinical experience. The hypothesis has been raised that the occurrence of severe disease may relate to the emergence of a variant form of the parasite. To test this hypothesis, we conducted a retrospective cohort study of P. vivax strains in the State of Amazonas. We determined nucleic acid sequences of segments of three genes, the 18S SSUrRNA Type A gene, the circumsporozoite surface protein (CSP) gene and the MSP-1 gene. Sequences were determined for parasites infecting 11 hospitalized (Inpatients) and 21 non-hospitalized (Outpatients) patients. We observed two common polymorphisms in the 18S SSUrRNA Type A gene; a thymidine (T)/adenine (A) polymorphism at residue 117 was significantly more common in the Inpatient group (p<0.05). Types of variation in the CSP gene included the numbers of repeat nonapeptide segments, alanine/aspartic acid polymorphism at position 5 of the nonapeptide repeat, and sporadic mutations. Alanine was more common as the fifth residue of the nonapeptide repeat in Inpatients and in strains causing second infections (both, p<0.05). Synonymous substitutions of the common repeat sequence occurred frequently in codons 1, 2, and 7, while the mutations at codon 5 were always non-synonymous, indicating that variation at codon 5 reflected selective pressure. Among MSP-1 gene sequences, recombination among progenitor strains, related to the Salvador I and Belém strains, was the main source of diversity. Phylogenetic analyses that incorporated sequence data for all three genes tested did not reveal clustering of sequences from inpatients. Our data do not affirm that the hypothesis that severe P. vivax disease in Amazonas is related to emergence of a new variant, but do suggest that variation in the fifth position of the CSP gene nonapeptide repeat may relate to disease manifestations.

Malaria diagnosis and hospitalization trends, Brazil.

We focused on rates of malaria in the state of Amazonas and city of Manaus, Brazil. Plasmodium vivax accounted for an increased number and rate of hospital admissions, while P. falciparum cases decreased. Our observations on malaria epidemiology suggest that the increased hospitalization rate could be due to increased severity of P. vivax infections.

Plasmodium-infected Anopheles mosquitoes collected in Virginia and Maryland following local transmission of Plasmodium vivax malaria in Loudoun County, Virginia.

Two recent outbreaks of locally acquired, mosquito-transmitted malaria in Virginia in 1998 and 2002 demonstrate the continued risk of endemic mosquito-transmitted malaria in heavily populated areas of the eastern United States. Increasing immigration, growth in global travel, and the presence of competent anopheline vectors throughout the eastern United States contribute to the increasing risk of malaria importation and transmission. On August 23 and 25, 2002, Plasmodium vivax malaria was diagnosed in 2 teenagers in Loudoun County, Virginia. The Centers for Disease Control and Prevention (CDC) deemed these cases to be locally acquired because of the lack of risk factors for malaria, such as international travel, blood transfusion, organ transplantation, or needle sharing. The patients lived approximately 0.5 mi apart; however, 1 patient reported numerous visits to friends who lived directly across the street from the other patient. Two Anopheles quadrimaculatus s.l. female pools collected in Loudoun County, Virginia, and 1 An. punctipennis female pool collected in Fairfax County, Virginia, tested positive for P. vivax 210 with the VecTest panel assay and enzyme-linked immunosorbent assay (ELISA). In addition, 2 An. quadrimaculatus s.l. female pools collected in Montgomery, Maryland, tested positive for P. vivax 210. The CDC confirmed these initial results with the circumsporozoite ELISA. The authors believe that this is the 1st demonstration of Plasmodium-infected mosquitoes collected in association with locally acquired human malaria in the United States since the current national malaria surveillance system began in 1957.

Use of polymerase chain reaction technique to confirm VecTest screening results in Plasmodium falciparum and Plasmodium vivax VK 210 laboratory-infected Anopheles stephensi mosquitoes.

We evaluated polymerase chain reaction (PCR) to confirm immunoassays for malaria parasites in mosquito pools after a failure to detect malaria with PCR during an outbreak in which pools tested positive using VecTest and enzyme-linked immunosorbent assay (ELISA). We combined VecTest, ELISA, and PCR to detect Plasmodium falciparum and Plasmodium vivax VK 210. Each mosquito pool, prepared in triplicate, consisted of 1 exposed Anopheles stephensi and up to 9 unfed mosquitoes. The results of VecTest and ELISA were concordant. DNA from a subset of the pools, 1 representative of each ratio of infected to uninfected mosquitoes, was extracted and used as template in PCR. All P. vivax pools were PCR positive but some needed additional processing for removal of apparent inhibitors before positive results were obtained. One of the pools selected for P. falciparum was negative by PCR, probably because of losses or contamination during DNA extraction; 2 remaining pools at this ratio were PCR positive. Testing pools by VecTest, ELISA, and PCR is feasible, and PCR is useful for confirmation of immunoassays. An additional step might be needed to remove potential inhibitors from pools prior to PCR.