Evaluation of commercially available preservatives for laboratory detection of helminths and protozoa in human fecal specimens.

Formalin and mercuric chloride-based low-viscosity polyvinyl alcohol (LV-PVA) are widely used by most diagnostic parasitology laboratories for preservation of helminth eggs and protozoan cysts and trophozoites in fecal specimens. Concerns about the toxicity of formalin and the difficulty of disposal of LV-PVA are powerful incentives to use alternate preservatives. Such alternatives have been marketed by several companies and are often presented as one-vial, non-mercuric chloride fixatives that aim at performing the same role as formalin and PVA combined. We compared five, one-vial commercial preservatives, two from Meridian Diagnostics, Inc. (Ecofix and sodium acetate-acetic acid-formalin), and one each from Scientific Device Laboratories, Inc. (Parasafe), Alpha Tec Systems, Inc. (Proto-fix), and Streck Laboratories, Inc. (STF), with 10% formalin and LV-PVA. Fecal specimens obtained from patients in a Brazilian hospital were aliquoted within 12 h of collection into the seven preservatives mentioned above and were processed after 1 month at the Centers for Disease Control and Prevention. Direct and concentrated permanent smears as well as concentrates for 20 positive specimens (a total of 259 processed samples) were prepared, stained according to the manufacturers' instructions, examined, and graded. Positive specimens contained one or more parasites with stages consisting of eggs, larvae, cysts, and a few trophozoites of Giardia intestinalis. Criteria for assessment of the preservatives included the quality of the diagnostic characteristics of helminth eggs, protozoan cysts, and trophozoites, ease of use, and cost. Acceptable alternatives to formalin for wet preparations were found. Ecofix was found to be comparable to the traditional "gold standard" LV-PVA for the visualization of protozoa in permanent stained smears. This study suggests that more acceptable alternatives to the traditional formalin and LV-PVA exist.

Prevalence of malaria parasitemia and accuracy of microscopic diagnosis in Haiti, October 1995.

In October 1995 the Ministry of Public Health and Population in Haiti surveyed 42 health facilities for the prevalence and distribution of malaria infection. They examined 1,803 peripheral blood smears from patients with suspected malaria; the overall slide positivity rate was 4.0% (range, 0.0% to 14.3%). The rate was lowest among 1- to 4-year-old children (1.6%) and highest among persons aged 15 and older (5.5%). Clinical and microscopic diagnoses of malaria were unreliable; the overall sensitivity of microscopic diagnosis was 83.6%, specificity was 88.6%, and the predictive value of a positive slide was 22.2%. Microscopic diagnoses need to be improved, and adequate surveillance must be reestablished to identify areas where transmission is most intense. The generally low level of malaria is encouraging and suggests that intensified control efforts targeted to the areas of highest prevalence could further diminish the effect of malaria in Haiti.

Antibodies to the ring-infected erythrocyte surface antigen of Plasmodium falciparum elicited by infection with Plasmodium malariae.

The ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum (RESA-P), found in the membrane of erythrocytes infected with young asexual stages of P. falciparum, is a promising vaccine candidate. Antibodies to RESA-P were inducible by infection with another human malaria species, P. malariae. Of 298 serum samples from inhabitants of three isolated localities in Peru where P. vivax and P. malariae were endemic and P. falciparum had never been reported, 26% had anti-RESA-P antibodies as evidenced by a modified immunofluorescent-antibody assay and confirmed by Western blot (immunoblot) analysis. These seroepidemiologic observations were corroborated by the fact that of six chimpanzees infected with P. malariae, three developed anti-RESA-P antibodies after infection. The modified immunofluorescent-antibody-reactive antibodies, purified by adsorption and elution on monolayers of glutaraldehyde-fixed and air-dried P. falciparum-infected erythrocytes, reacted in an immunofluorescent-antibody assay with both parasite structures and erythrocyte membrane in P. falciparum antigen preparations, but only with parasite structures in P. malariae antigen preparations. This serologic cross-reactivity between P. falciparum and P. malariae is of interest in view of the importance of RESA-P as a vaccine candidate and because the two species are coendemic in many areas.

Antibodies to Pf155, a major antigen of Plasmodium falciparum: seroepidemiological studies in Haiti.

The presence of malaria parasites and the serological antibody responses against whole Plasmodium falciparum and the Pf155 antigen were studied in the population of a small rural locality in Haiti in December 1985. Only 7 (1.5%) of the individuals were found to be infected with P. falciparum, the only species observed. Antibodies to P. falciparum were detected in an ELISA in 38.2% of the sera, the positivity rates being age-related. Anti-Pf155 antibodies were detected in 12.5% and 13.6% of individuals by two different techniques used. The anti-Pf155 positivity rates increased only after 25 years of age. No trends were detected for a clear-cut protective value of Pf155 antibodies against clinical malaria and further longitudinally conducted field surveys are needed to satisfactorily assess the potential protective effect of Pf155 antibodies.

Monitoring of chloroquine sensitivity of Plasmodium falciparum in Haiti, 1981-1983.

Between 1981 and 1983, in vivo and in vitro studies were conducted in Haiti to assess the responsiveness of Plasmodium falciparum to chloroquine. The standard tests successfully performed included 92 WHO standardized in vivo field tests and 160 in vitro tests (64 macrotests, 33 microtests, and 63 48-hr tests). No clearcut evidence of chloroquine resistance was detected. In 3 in vivo and 5 in vitro tests, a decreased susceptibility to the drug was suggested, but these isolated findings failed to be corroborated by parallel alternate tests. In addition, during the initial trial of an alternate monitoring system, 339 simplified 7-day in vivo tests were successfully performed, with no suggestion of resistance detected. This simplified 7-day in vivo test potentially represents an efficient low cost method for monitoring drug resistance in many developing countries.

Development of an in vitro microtest for determining the susceptibility of Plasmodium falciparum to sulfadoxine-pyrimethamine: laboratory investigations and field studies in Port-au-Prince, Haiti.

An in vitro microtest for assessing the susceptibility of Plasmodium falciparum to sulfadoxine-pyrimethamine (S-P) was developed following WHO guidelines. Paraaminobenzoic acid and folic acid were depleted in the culture medium used, the test wells were predosed with sulfadoxine and pyrimethamine at a constant ratio of 80:1, and the parasites were incubated for 48 hours. Optimum parasite multiplication was obtained with a 2% erythrocyte suspension in medium supplemented with 12% serum. During in vitro studies with laboratory-adapted isolates, response patterns were obtained which distinguished 3 isolates with documented in vivo sensitivity to S-P from 2 isolates with documented in vivo resistance to S-P. In addition, among the three S-P-sensitive isolates, one isolate that was pyrimethamine-resistant in vitro had a higher S-P inhibitory endpoint than 2 isolates that were pyrimethamine-sensitive in vitro. The S-P microtest was further evaluated in combined in vivo and in vitro studies in Port-au-Prince, Haiti. Twenty-six patients infected with P. falciparum were treated with standard doses of S-P, resulting in prompt clearance of parasitaemia, with no recurrence in the 24 patients who completed a 28-day follow-up period. Parallel in vitro tests with pyrimethamine alone showed 3 pyrimethamine-resistant isolates out of 22 successful tests on the patients' blood samples. In 23 successful S-P tests, the known in vivo S-P-sensitive parasites were inhibited at S-P concentrations that were generally lower for in vitro pyrimethamine-sensitive isolates than for in vitro pyrimethamine-resistant ones.

Plasmodium falciparum in Haiti: susceptibility to pyrimethamine and sulfadoxine-pyrimethamine.

Eighteen patients with Plasmodium falciparum infection were studied in Port-au-Prince, Haiti, to monitor the response of the malaria parasite to sulfadoxine-pyrimethamine. In all infections the parasitaemia was cleared rapidly following treatment with standard dose of the drug combination; no recrudescence was observed during follow-up periods of 1 week (4 patients) and 4 weeks (14 patients). Parallel in vitro tests indicated that 5 of the 16 isolates successfully tested were resistant to pyrimethamine alone.

Studies on the Indochina I/CDC strain of Plasmodium falciparum in Colombian and Bolivian Aotus monkeys and different anophelines.

The Indochina I/CDC strain of Plasmodium falciparum was isolated from a physician returning to the United States after working in the refugee camps along the Thailand-Kampuchean border. The strain was established in splenectomized Aotus monkeys from Colombia after being grown in vitro for 50 days. During the first three passages in Colombian monkeys, the parasites were not infective to Bolivian Aotus monkeys. After six intervening passages in Saimiri sciureus monkeys, the parasites produced high parasitemias in both Colombian and Bolivian Aotus, but gametocytes were no longer produced. Mosquito infections were obtained only during the first three passages in the Colombian monkeys. The most susceptible mosquito was Anopheles freeborni, followed by An. dirus, An. stephensi, An. maculatus, An. culicifacies, and, rarely, An. gambiae. Sporozoites were found in the salivary glands of the An. freeborni, An. dirus, An. stephensi, and An. maculatus.

Chloroquine susceptibility of Plasmodium falciparum in Haiti.

In January and February 1982, in vivo and in vitro studies of the chloroquine sensitivity of Plasmodium falciparum were conducted in Port-au-Prince, Haiti. Of 19 infections tested in vivo using the WHO extended test, all but one were susceptible to the drug; the remaining case showed a recurrence of parasitaemia on day 28. Of the 19 corresponding 48-hour in vitro tests, 16 provided interpretable results; 12 tests demonstrated sensitivity to the drug, while in the remaining 4, parasite multiplication was inhibited only at drug concentrations higher than that previously accepted as indicative of sensitivity. (These four included the isolate from the patient who had a recurrence of parasitaemia on day 28.) While these results provide no absolute demonstration of chloroquine resistance, they underline the need for close monitoring of the susceptibility to chloroquine of P. falciparum in Haiti.

Assessment of chloroquine sensitivity of Plasmodium falciparum in Choluteca, Honduras.

During an outbreak of urban malaria in Choluteca, Honduras, the response of local isolates of Plasmodium falciparum to chloroquine was assessed. The 7-day WHO alternative standard field test was used together with three in vitro tests: the Rieckmann macro- and micromethods and a new 48-hour test which underwent its first field trial in this study. No chloroquine resistance was found in in vivo tests in 10 patients or in the in vitro tests on blood samples from 6 patients.