Bernard L. Nahlen

Polymorphism in the gene encoding the apical membrane antigen-1 (AMA-1) of Plasmodium falciparum. X. Asembo Bay Cohort Project

We have investigated the genetic diversity of the gene encoding the apical membrane antigen-1 (AMA-1) in natural populations of Plasmodium falciparum from western Kenya and compared it with parasite populations from other geographic regions. A total of 28 complete sequences from Kenya, Thailand, India, and Venezuela field isolates were obtained. The genetic polymorphism is not evenly distributed across the gene, which is in agreement with the pattern reported in earlier studies. The alleles from Kenya exhibit 20 and 30% more polymorphism than that found in Southeast Asia and Venezuelan alleles, respectively. Based on the gene genealogies derived from sequencing data, no evidence for allele families was found. We have found evidence supporting limited gene flow between the parasite populations, specifically, between the Southeast Asian and Venezuelan isolates; however, no alleles could be linked to a specific geographic region. This study reveals that positive natural selection is an important factor in the maintenance of genetic diversity for AMA-1. We did not find conclusive evidence indicating intragenic recombination is important in the generation of the AMA-1 allelic diversity. The study provides information on the genetic diversity of the AMA-1 gene that would be useful in vaccine development and testing, as well as in assessing factors that are involved in the generation and maintenance of the genetic diversity in P. falciparum.

A simple perfusion technique for isolation of maternal intervillous blood mononuclear cells from human placentae

A noninvasive perfusion method for the recovery of maternal placental (intervillous) blood for use in immunologic assays is described. 60% of the perfused blood samples tested for fetal red blood cell (RBC) contamination were found to be pure maternal blood; in the remainder, fetal RBC contamination, with a single exception, was less than 6%. The intervillous mononuclear cells (IVBMC) isolated from this blood were of predominantly maternal origin as demonstrated by a polymerase chain reaction-based DNA typing technique. The number of IVBMC obtained was within the range of 9 to 55 X 10(6) cells. Phenotypic analysis of IVBMC surface antigens revealed that 61% of the cells were CD3 + T-cells and 18% were CD19 + B-cells. The CD4 + and CD8 + T-lymphocyte subsets accounted for 28 and 26% of the IVBMC, respectively. The IVBMC were functionally competent as evidenced by in vitro lymphoproliferation and cytokine production in response to mitogen and PPD stimulation. This technique allows for rapid and safe isolation of large numbers of IVBMC which are functionally active up to 12 h post-delivery, thus representing a significant improvement over previously described methods. It should facilitate more vigorous research in the study of uteroplacental immunity and infectious disease research, particularly in field settings where sample collection and laboratory facilities are distant.

Immunity to Placental Malaria. II. Placental Antigen—Specific Cytokine Responses Are Impaired in Human Immunodeficiency Virus—Infected Women

An association was demonstrated recently between elevated in vitro production of interferon (IFN)-gamma by intervillous blood mononuclear cells (IVBMCs) and protection against placental malaria (PM). Because human immunodeficiency virus (HIV)-infected pregnant women have increased susceptibility to PM, loss of the IFN-gamma response in these women may impair their ability to control PM. Measurement of cytokines in culture supernatants by ELISA revealed that IFN-gamma responses by HIV-positive IVBMCs were impaired, especially after malarial antigen stimulation. Interleukin (IL)-4 and IL-10 responses also were reduced in HIV-positive persons, the latter more so in HIV-positive, PM-positive persons. In contrast, tumor necrosis factor-alpha production generally was enhanced in PM-positive and HIV-positive persons. Overall, cytokine production was reduced in HIV-positive persons with CD4 T cell counts <500/microL, particularly in response to malarial antigen. Thus, HIV-mediated cytokine dysregulation and impairment of the protective IFN-gamma response may contribute to the increased susceptibility of HIV-positive pregnant women to malaria.

LACK OF EFFICACY OF PYRIMETHAMINE PROPHYLAXIS IN PREGNANT NIGERIAN WOMEN

To evaluate the efficacy of pyrimethamine on the blood stage (suppressive prophylaxis) and liver stage (causal prophylaxis) of Plasmodium falciparum in pregnant women, in vivo and in vitro field studies were conducted in Ilorin, Nigeria, from Jan 1 to June 30, 1988. For pregnant women with P falciparum infections who received 25 mg of pyrimethamine weekly for suppressive prophylaxis, 67% (59/88) of in vivo and 60% (6/10) of in vitro tests showed pyrimethamine resistance. A second group of parasitaemic and parasite-free pregnant women was enrolled to evaluate the efficacy of pyrimethamine as a primary tissue schizonticide; after receiving a curative dose of chloroquine (25 mg/kg), half the women were given 25 mg of pyrimethamine weekly and half received no prophylaxis. Parasitologic failure rates did not differ between the pyrimethamine-treated (8/34) and the control (11/37) groups during the 16-week follow-up. Thus, pyrimethamine is not effective for suppressive or causal prophylaxis in pregnant women in Ilorin.

A rapid assessment approach for public health decision-making related to the prevention of malaria during pregnancy

OBJECTIVE To develop a rapid field assessment methodology to address the burden of malaria during pregnancy and the options for intervening within the existing antenatal care system in Kenya. METHODS Surveys consisting of questionnaires, sampling of blood for parasitaemia and anaemia, and birth outcome assessment were conducted in antenatal clinics, delivery units, and in the community in Kisumu and Mombasa, Kenya. FINDINGS The rates of maternal anaemia and severe anaemia, were, respectively, 79% and 8% in Kisumu, and 95% and 24% in Mombasa. The rates of placental parasitaemia were 27% and 24% and the rates of low birth weight were 18% and 24% in Kisumu and Mombasa, respectively. Women with placental parasitaemia had a higher incidence of low birth weight compared with women without placental parasitaemia in both Kisumu (28% vs 16%, P=0.004) and Mombasa (42% vs 20%, P=0.004). A total of 95% and 98% of women in Kisumu and Mombasa, respectively, reported attending an antenatal clinic during their previous pregnancy. CONCLUSION This methodology can be used by ministries of health to collect data for decision-making regarding malaria control during pregnancy; it can also provide a baseline measurement on which to evaluate subsequent interventions.