Performance of the OptiMAL assay for detection and identification of malaria infections in asymptomatic residents of Irian Jaya, Indonesia.

The OptiMAL assay, a new immunochromatographic "dipstick" test for malaria based on detection of Plasmodium lactate dehydrogenase (pLDH), is purported to detect infections of approximately 200 parasites/microL of blood and to differentiate between Plasmodium falciparum and non-P. falciparum. We evaluated OptiMAL performance by comparing the test strip interpretations of two independent readers with consensus results obtained independently by expert malaria microscopists. Unbiased measures of sensitivity were derived by applying the OptiMAL test for detection and differentiation of light, asymptomatic infections by P. falciparum and Plasmodium vivax. OptiMAL readings were separated in time to determine whether the reaction signal was stable. Microscopy identified infections in 225 of 505 individuals screened; those with P. falciparum (n = 170) averaged 354 asexual forms/microL and P. vivax/Plasmodium malariae (n = 112) averaged 216 asexual forms/microL of blood. Concordance between OptiMAL and microscopy was 81% and 78% by the two independent readings. The assay's sensitivity for detection of any malaria species was 60.4% and 70.2% respectively and specificity was 97% and 89%. Most cases identified by microscopy as P. falciparum were graded as negative or non-falciparum by both OptiMAL readers. OptiMAL false negatives as well as misidentifications were related to low parasitemias (< 500/microL). The OptiMAL assay demonstrated 88-92% sensitivity for detecting infections of 500-1,000 parasites/microL, a range covering the mean parasitemia of primary symptomatic P. falciparum infections in malaria-naïve Indonesian transmigrants. This device was markedly less sensitive than expert microscopy for discriminating between malaria species and is presently unsuited for use as an epidemiological screening tool. The OptiMAL assay is not approved for diagnostic use but is commercially available for research purposes only.

Lymphocyte response to tetanus toxin T-cell epitopes: effects of tetanus vaccination and concurrent malaria prophylaxis.

Synthesized T-cell epitopes of tetanus toxin are universally immunogenic and serve to enhance immune response when they are used as vaccine carriers of B-cell epitopes. The immunogenicity of the P2, P30, and P2P30 T-cell epitopes of tetanus toxin and whole tetanus toxoid (TT) was evaluated by in vitro proliferation assay of lymphocytes from men with no history of tetanus vaccination who were enrolled in a malaria prophylaxis trial. The enhancement of immune response by tetanus vaccination (Td) and possible antagonism by the antimalarial drugs, was measured by pre- and post-Td comparisons within and between immunized prophylaxis groups (primaquine, chloroquine, placebo) and a nonimmunized control group. Constructs demonstrated low immunogenicity relative to TT in all groups. Relative to both control and its own baseline, the immunized primaquine prophylaxis group was distinct in demonstrating significantly increased proliferation against all three subunits and at both high (30 microg ml(-1)) and low (3 microg ml(-1)) concentrations. Immunization elicited significantly increased proliferation responses by placebo and chloroquine prophylaxis groups against only the P2P30 construct. Despite these significant post-Td changes, a low concentration of TT 0.1 microg ml(-1)) stimulated proliferation 7-10 times over that induced by the greatest concentration of the constructs.

Humoral immune response to tetanus-diphtheria vaccine given during extended use of chloroquine or primaquine malaria chemoprophylaxis.

Immune suppression resulting from prolonged chemoprophylaxis and potential drug-vaccine interaction were investigated within the context of a randomized placebo-controlled trial that compared daily primaquine or weekly chloroquine administration for malaria prevention. After 11 months of prophylaxis, adult male subjects received a tetanus-diphtheria (Td) vaccination. Prophylaxis continued 4 weeks longer. Anti-tetanus and anti-diphtheria antibody levels were measured by ELISA at baseline and at 1, 3, 7, and 14 months after Td vaccination. All groups were comparable at baseline. Immunization triggered significant increases in anti-tetanus and anti-diphtheria IgG levels over each group's pre-Td baseline levels and those of an unvaccinated control group. Geometric mean anti-tetanus titers (GMTs) in the primaquine group were significantly higher than those of the placebo group at 1, 3, and 14 months. Anti-tetanus GMTs in placebo and chloroquine groups declined over 14 months to levels comparable to those of unvaccinated controls, but levels in the primaquine group remained significantly higher than in controls.

Lymphocyte response to tetanus toxoid among Indonesian men immunized with tetanus-diphtheria during extended chloroquine or primaquine prophylaxis.

Immune suppression, a potential side effect of long-term chemoprophylaxis, was evaluated as part of a randomized, placebo-controlled trial that compared daily primaquine against weekly chloroquine for malaria prevention. In the last month of the year-long trial, baseline in vitro lymphoproliferative responses to tetanus toxoid were measured, and a tetanus-diphtheria (Td) immunization was administered. Proliferative responses to tetanus toxoid in each Td-immunized group increased significantly over pre-Td baselines and those of the unvaccinated control. Highest initial responses were measured in the primaquine group. The proportion of responders and the magnitude of proliferation was consistently low in the chloroquine group, and end point responses in this group were significantly below those of the placebo. These results suggest that the development and duration of the cellular response to tetanus immunization was impaired by long-term weekly chloroquine prophylaxis, while daily primaquine prophylaxis over the same time period had no inhibitory effect.

Survey of in vivo sensitivity to chloroquine by Plasmodium falciparum and P. vivax in Lombok, Indonesia.

A malariometric survey was conducted in 14 villages of Sekotong district, in Lombok, Indonesia during October 1994. Point prevalence of malaria ranged from 0% to 15% in the surveyed villages, averaging 6% overall, and Plasmodium falciparum accounted for 63% of the infections. Forty-nine patients with uncomplicated malaria and parasite counts ranging from 40 to 10,800 asexual forms/microliter were enrolled in a 28-day in vivo test of chloroquine sensitivity. All subjects received a supervised therapeutic regimen of chloroquine (25 mg base/kg over a 48-hr period) and parasitemia and symptoms were closely monitored for 28 days. Asexual parasites were eliminated within four days in the 29 P. falciparum and 20 P. vivax study patients enrolled. The cumulative incidence of therapeutic failure (recurrent symptomatic parasitemia) among P. falciparum cases at days 7, 14, and 28 was 7%, 10%, and 14% (4 of 29), respectively. However in all four cases, parasitemias recurred against chloroquine blood levels below the minimally effective concentration (MEC) of 200 ng/ml and do not confirm chloroquine resistance. All 20 P. vivax parasitemias were sensitive to chloroquine and the blood remained clear, with the exception of one case in which an asymptomatic parasitemia appeared on day 28. Parasitemias by P. falciparum and P. vivax that were observed before supervised therapy, but in the presence of whole blood chloroquine above normally suppressive MEC levels, suggest resistance to suppressive or prophylactic regimens of chloroquine.

Comparative performance of the ParaSight F test for detection of Plasmodium falciparum in malaria-immune and nonimmune populations in Irian Jaya, Indonesia.

A comparison was made of the performance of the ParaSight F test (F test) for detection of Plasmodium falciparum in blood from malaria-immune (410 native Irianese) and nonimmune (369 new transmigrants) populations in Irian Jaya, Indonesia, where malaria is hyperendemic and all four species of human malaria occur. There were highly significant differences between populations in the sensitivity (Irianese, 60% versus transmigrants, 84%; P < 0.001) and specificity (Irianese, 97% versus transmigrants, 84%; P < 0.001) of the F test. The test had comparably high levels of sensitivity for Irianese children aged < or = 10 years, both age groups of transmigrants (76-85%), but low sensitivity for Irianese aged > 10 years (40%), among whom only 7% of parasitaemias < 120 per microliter and 69% of those > 120 per microliter were detected. Specificity was comparably high for transmigrant children aged < or = 10 years and both age groups of Irianese (93-98%). The low specificity for transmigrants aged > 10 years (79%) was due to a preponderance of false positives, frequently identified by microscopy as P. vivax. The results suggest that comparison based on microscopy underestimated the performance of the ParaSight F test and that malaria immune status, irrespective of P. falciparum density, may influence the test's sensitivity.

PIP: The ParaSight F test uses a nonmicroscopic dipstick approach to the rapid detection of Plasmodium falciparum in blood. The performance of this test was assessed in serum samples collected in Irian Jaya, Indonesia, from 410 native Irianese (malaria-immune) and 369 new transmigrants (nonimmune). Of particular interest was the capability of the F test to detect P. falciparum prevalence among children, whose immunity is less than that of adults. There were highly significant differences by population in the F test's sensitivity (60% for Irianese vs. 84% for transmigrants) and specificity (97% for Irianese vs. 84% for transmigrants). The test had high sensitivity levels (76-85%) for Irianese children 10 years of age and under and both child and adult transmigrants, but low sensitivity (40%) for Irianese over 10 years of age. Specificity was comparably high (93-98%) for transmigrant children and both age groups of Irianese. The low specificity (79%) for transmigrants over 10 years of age reflected a preponderance of false positives, frequently identified by microscopy as P. vivax. These findings suggest that microscopy comparisons underestimate the performance of the ParaSight F test and that malaria immune status, regardless of P. falciparum density, may influence the test's sensitivity.

Lymphocyte proliferative response and subset profiles during extended periods of chloroquine or primaquine prophylaxis.

Immune suppression and disturbances of normal leukocyte populations are side effects attributed to many antimalarial drugs and were concerns during a recent year-long placebo-controlled trial that compared daily primaquine (0.5 mg of base per kg of body weight per day) with weekly chloroquine (300 mg of base one time per week) for malaria prophylaxis. The study took place in Irian Jaya, Indonesia, from July 1994 to August 1995 and enrolled 129 Javanese men with normal glucose-6-phosphate dehydrogenase function. Tests for lymphocyte function and subset composition were conducted blindly on a cross-section of subjects during weeks 10 (n = 42) and 48 (n = 72) of supervised prophylaxis. Lymphocyte function, measured as the proliferative response of peripheral blood mononuclear cells to a panel of mitogens (pokeweed mitogen, phytohemagglutinin, and concanavalin A) and antigens (purified protein derivative of Mycobacterium tuberculosis and Clostridium tetani toxoid) and expressed as a stimulation index, allowed for statistical comparison between groups and sampling times. The lymphocyte subset composition for each group and time point was based on flow cytometry profiling, and the results were expressed as the mean percentages of CD3 (total T cells), CD19 (total B cells), CD4+ (T-helper and inducer cells), and CD8+ (T suppressor and cytotoxic cells), CD3/CD16+ CD56 (natural killer cells), CD3/anti-HLA-DR (activated T cells) cells and the CD4+/CD8+ ratios. Lymphocyte stimulation indices were statistically comparable among the placebo, primaquine, and chloroquine groups at both time points, although the primaquine group was distinguished by having repeatedly greater proportions of subjects with high ( > 3.0) stimulation indices. The lymphocyte subset profiles of these groups at both time points were also similar and undistorted relative to those of healthy reference populations matched for age, sex, and ethnicity. The results provide quantitative support for the safety of daily primaquine prophylaxis.