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.

Malaria, intestinal parasites, and schistosomiasis among Barawan Somali refugees resettling to the United States: a strategy to reduce morbidity and decrease the risk of imported infections.

In 1997, enhanced health assessments were performed for 390 (10%) of approximately 4,000 Barawan refugees resettling to the United States. Of the refugees who received enhanced assessments, 26 (7%) had malaria parasitemia and 128 (38%) had intestinal parasites, while only 2 (2%) had Schistosoma haematobium eggs in the urine. Mass therapy for malaria (a single oral dose of 25 mg/kg of sulfadoxine-pyrimethamine) was given to all Barawan refugees 1-2 days before resettlement. Refugees >2 years of age and nonpregnant women received a single oral dose of 600 mg albendazole for intestinal parasite therapy. If mass therapy had not been provided, upon arrival in the United States an estimated 280 (7%) refugees would have had malaria infections and 1,500 (38%) would have had intestinal parasites. We conclude that enhanced health assessments provided rapid on-site assessment of parasite prevalence and helped decrease morbidity among Barawan refugees, as well as, the risk of imported infections.

Adaptation of a strain of Plasmodium vivax from Mauritania to New World monkeys and anopheline mosquitoes.

A strain of Plasmodium vivax from Mauritania was adapted to develop in Aotus lemurinus griseimembra, Aotus nancymai, Saimiri boliviensis, and hybrid Aotus monkeys. Infections were induced via the inoculation of sporozoites dissected from the salivary glands of Anopheles gambiae, Anopheles freeborni, and Anopheles stephensi mosquitoes or the intravenous passage of infected erythrocytes. Infections in 3 A. lemurinus griseimembra monkeys readily infected mosquitoes. Four lines of the Mauritania parasites have been stored frozen for further reference.

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.

Evaluation of national malaria control programmes in Africa.

Evaluation is an essential management tool for the improvement of public health programmes or projects. As malaria morbidity and mortality continue to increase in most countries in Africa, international agencies and malaria control programme managers have identified the strengthening of programme evaluation as an important strategy for improving the efficiency and effectiveness of malaria control programmes. Managers can develop an evaluation strategy only after they have defined programme objectives and planned specific programme activities. Indicators should be directly related to programme objectives and should be selected on the basis of the following criteria: their validity; reliability; ability to detect change within a reasonable time period and as a result of successful programme implementation; ability to be interpreted; and usefulness in guiding programme change. Only those indicators that can be measured with available programme resources should be selected. Managers will also need to identify the sources of indicator data and to determine how often each indicator will be measured. Programme managers should develop criteria or indicators for the following: programme policies and plans; the process of programme implementation; the outcomes of malaria control interventions in disease management and prevention; and programme impact in terms of reductions in malaria-related mortality and morbidity. Key issues related to the management of evaluation activities within a national programme include the need to begin with available resources and build incrementally; to explore options for administering evaluation activities; to select, train and supervise staff who carry out evaluation activities; to develop quality control strategies; and to ensure that data are managed and communicated in ways that support effective programme decision-making. For evaluation to lead to improvements in malaria control programmes it must be clearly defined as a part of the programme management process. Programme managers should lead this developmental process, ensuring that evaluation methods produce the information they need to monitor and improve their programmes at reasonable cost.

Plasmodium falciparum-associated anemia in children at a large urban hospital in Zaire.

Chloroquine-resistant Plasmodium falciparum malaria and human virus (HIV) infection through blood transfusions used to treat malaria-associated anemia are causes of increasing morbidity and mortality among children in Africa. To evaluate the role of malaria and other risk factors for pediatric anemia, we conducted a study of children brought to the emergency ward of a large urban hospital in Kinshasa, Zaire. A total of 748 children ages six through 59 months were enrolled; 318 (43%) children were anemic (hematocrit < 33%), including 74 (10%) who were severely anemic (hematocrit < 20%). Plasmodium falciparum parasites were detected in 166 children (22%); hematocrits for these children (mean 25.8%) were significantly lower than for aparasitemic children (mean 33.7%; P < 10(-6)). Fever with splenomegaly (odds ratio [OR] = 6.5, P = 0.02), parasitemia (OR = 3.5, P < 0.001), lower socioeconomic status (OR = 2.0, P = 0.004), and malnutrition (OR = 1.8, P = 0.06) were independently associated with anemia in a multivariate model. Recent antimalarial therapy was also associated with a lower hematocrit, suggesting that chloroquine may have aggravated the anemia. A reassessment of the effectiveness of strategies to diagnose and treat malaria and malnutrition is necessary to decrease the high prevalence of anemia and the resultant high rate of blood transfusions in areas endemic for malaria and HIV.

Plasmodium cynomolgi: immunization of a rhesus monkey with exoerythrocytic stages cultured in autologous hepatocytes.

To investigate the immune response to exoerythrocytic stages of malaria parasites, a rhesus monkey was immunized with autologous primary hepatocyte cultures infected with 7-day-old liver stage parasites of Plasmodium cynomolgi. A primary antibody response against EE stage antigens was obtained, and boosted after injection of homologous viable sporozoites. Antibodies directed against sporozoites and blood stages were also detected. The polyvalent immune response observed demonstrates the antigenicity of the liver stages and suggests their involvement in the general immune response against malaria.

Trends and risk factors for HIV-1 seropositivity among outpatient children, Kinshasa, Zaire.

To investigate recent trends in pediatric HIV-1 infection and the early impact of a blood screening program begun in one hospital in 1987 in Kinshasa, Zaire, we evaluated 1110 consecutive children seen in the pediatric emergency ward of the city's largest hospital in November 1988. The HIV-1 seroprevalence was 5.0%, not significantly higher than the rate of 3.8% found in 1986 (P = 0.2). The seropositivity rate was bimodally distributed; children less than 6 months of age had a higher rate (12.6%) than children 6-11 months old (1.9%; OR = 7.6; P less than 0.0001) and children 1-13 years old (4.1%; OR = 3.4; P less than 0.0001). Seropositive children greater than or equal to 1 year of age were more likely than seronegative children to be anemic and to have signs of malnutrition. A previous blood transfusion was associated with HIV-1 seropositivity among children greater than or equal to 1 year of age (OR = 5.4, P less than 0.0005), but not among younger children. Fifty-two per cent of seropositive children greater than or equal to 1 year of age received a transfusion (etiological fraction = 42%). The association with seropositivity was higher for those who had received a transfusion before 1987 than for those who had received a transfusion since 1987 (OR = 4.8, P = 0.01). These findings suggest a relatively stable, high pediatric HIV-1 seroprevalence in Kinshasa and a decreased but continued risk of transfusions. Expansion of currently limited blood transfusion screening programs, and the development of new strategies for limiting transfusions and preventing severe anemia, are needed.

PIP: To investigate recent trends in pediatric HIV-1 infection and the early impact of a blood screening program begun in 1 hospital in Kinshasa, Zaire, the authors evaluated 1110 consecutive children seen in the pediatric emergency ward of the city's largest hospital in November 1988. The HIV-1 seroprevalence was 5.0%, not significantly higher than the 3.8% rate found in 1986 (p=0.2). The seropositivity rate was bimodally distributed; children 6 months of age had a higher rate (12.6%) than children 6-11 months old (1.9%; OR+7.6; p0.0001) and children 1-13 years old (4.1%; OR+3.4; p0.0001). Seropositive children or= 1 year of age were more likely than seronegative children to be anemic and to have signs of malnutrition. A previous blood transfusion was associated with HIV-1 seropositivity among children or= 1 year of age (OR=5.4, p0.0005), but not among younger children. 52% of seropositive children or= 1 year of age had received a transfusion (etiological fraction=42%). The association with seropositivity was higher for those who had received a transfusion before 1987 than for those who received 1 since that time (OR=4.8, p=0.01). These findings suggest a relatively stable, high pediatric HIV-1 seroprevalence in Kinshasa and a decreased but continuous risk of transfusions. Expansion of currently limited blood transfusion screening programs and the development of new strategies for limiting transfusions and anemia prevention are necessary.

Use of non-human primate hepatocytes for in vitro study of the pre-erythrocytic stages of malaria parasites.

Methods were developed that allow invasion of sporozoites from simian malaria parasite species (Plasmodium cynomolgi, P. knowlesi, P. coatneyi, P. inui, P. gonderi, P. fragile) and development to schizont stages in rhesus and Saimiri monkey hepatocytes. The P. cynomolgi-rhesus monkey model was used to study inhibition of schizont development using monoclonal antibodies (MAbs) produced against the circumsporozoite (CS) protein of various strains and species of malaria parasites. Immunoelectron microscopy, using gold-labelled MAbs and cultured parasites, demonstrated that the CS protein persists in 7-day old liver stages of P. cynomolgi, but is not expressed at the surface of infected hepatocytes. A rhesus monkey was immunized with autologous hepatocytes (collected by biopsy) infected in vitro with liver stages of P. cynomolgi. This immunization elicited antibodies reacting with sporozoite, liver stage, and blood-stage parasites. In addition, human malaria parasites (P. falciparum, P. vivax, P. malariae) have been cultured in Saimiri or rhesus monkey hepatocytes. The P. vivax-Saimiri monkey model was used to study inhibition activity of sera from Saimiri monkeys experimentally immunized with recombinant P. vivax CS proteins. Post-immunization sera inhibited the parasite development, thus demonstrating the induction of antibodies effective against sporozoites. No relationship, however, was detected between in vitro inhibition and in vivo protection or antibody titres determined by ELISA or IFA.

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.

PIP: New studies on the suppressive and curative effects of the anti-malarial drug pyrimethamine in pregnant women from Ilorin, Nigeria showed both ineffective prophylaxis and suppression, and parasite resistance. The drug has been used in pregnant women because of its effectiveness in suppression of asexual forms of malaria infections due to Plasmodium falciparum, its long half life and its safety. 1st a group of 88 pregnant women infected with only P falciparum received 25 mg pyrimethamine weekly for 4 weeks and parasites were counted on blood smears. 67% retained parasites by Day 7, and 60% by Day 14. All were treated with curative doses of chloroquine. A 2nd group of 71 pregnant women were first treated for malaria parasites with 2 doses of chloroquine, 25 mg/kg, in 300 mg tablets, followed by weekly pyrimethamine 25 mg for 10 weeks. All subjects and controls were given iron and folic acid supplements to take daily. 24% developed parasitemia during the 10 weeks of the study, compared to 30% of the controls. The mean intervals to development of parasitemia, and the geometric mean parasite density in blood did not differ significantly. In 6 of 10 in vitro tests of parasite resistance to pyrimethamine, parasite growth was uninhibited, compared to 22 of 23 tests for resistance to chloroquine. In vivo and in vitro tests correlated well for pyrimethamine resistance. The results also indicated that primigravidae, who are more likely to harbor malaria parasites, were also more likely to fail in parasite suppression with pyrimethamine treatment. Thus pyrimethamine is not expected to reduce incidence of premature and low birth weight infants due to malaria in this area.

In-vitro exoerythrocytic development of Plasmodium cynomolgi bastianellii: inhibitory activity of monoclonal antibodies against sporozoites of different P. cynomolgi strains and of P. knowlesi.

The inhibitory effect of anti-sporozoite monoclonal antibodies (MoAb) on the in-vitro development of liver stages of Plasmodium cynomolgi bastianellii (NIH strain) was evaluated using primary cultures of rhesus monkey hepatocytes. MoAbs against the circumsporozoite proteins of five strains of P. cynomolgi (NIH, London, Gombak, Ceylon, Berok), and of P. knowlesi (H strain) were used. Incubation of sporozoites of P. cynomolgi bastianellii with the anti-NIH strain MoAbs entirely prevented liver-stage development; MoAbs produced against the other four strains had no apparent activity. The anti-P. knowlesi MoAbs had a partially inhibitory effect on parasite development. These functional studies complement previous immunological studies on P. cynomolgi strain specificity, and confirm the cross-reactivity observed previously between sporozoites of P. cynomolgi bastianellii and P. knowlesi (H strain).

Formulation of malaria treatment policy for children in Côte d'Ivoire as chloroquine resistant Plasmodium falciparum spreads into west Africa.

To develop a malaria treatment policy for children with Plasmodium falciparum, an in vivo and in vitro chloroquine (CQ) sensitivity study was conducted in Côte d'Ivoire in September 1986. The efficacy of a single dose of CQ (10 mg base kg-1, C10) was tested with assessment of subjects on Days 2 and 7 after treatment; 108 (99%) of 109 children were aparasitaemic on Day 7. Of 33 isolates of P. falciparum tested in vitro, two (6%) were resistant to CQ. Although C10 appeared effective clinically and parasitologically in Côte d'Ivoire, a treatment dose of 25 mg of CQ base kg-1 (C25), over three days, was recommended as first-line therapy for malaria. This was because in vivo CQ-resistance will soon spread into other West African countries including Côte d'Ivoire, the C25 dose still retains clinical effectiveness in most partially-immune persons living in areas with low-level chloroquine resistance, and alternate drugs are more expensive.

Intravenous quinine therapy of hospitalized children with Plasmodium falciparum malaria in Kinshasa, Zaire.

To examine the clinical and parasitologic efficacy of quinine, we studied 34 children (7 months-13 years old) with severe or moderately severe Plasmodium falciparum infections. Quinine 10 mg/kg every 8 hr for 3 days was administered, initially by intravenous infusion of quinine formate followed by oral quinine dihydrochloride when tolerated. Thirty-three of the 34 patients were clinically well and had negative malaria smears 7 days after the initiation of therapy; 1 child, who presented in coma, died 29 hr after enrollment. The mean fever clearance time was 44.1 hr, and the mean parasite clearance time was 59.6 hr. A mean peak quinine level of 9.7 ppm was attained after the second dose of quinine, and the minimum concentration was maintained at 5-7 ppm during the 2nd and 3rd hospital days. In vitro testing was conducted with parasites from 10 patients: 9 isolates were resistant to chloroquine, and inhibition of schizont development with quinine occurred at a concentration of 8-32 pmol/well.

In vitro activity of antimalarial compounds on the exoerythrocytic stages of Plasmodium cynomolgi and P. knowlesi.

Primary cultures of Macaca mulatta hepatocytes infected with sporozoites of Plasmodium knowlesi, P. cynomolgi (Cambodian strain), and P. cynomolgi bastianellii were exposed in vitro to 7 antimalarial compounds. The number of exoerythrocytic schizonts present after 4-7 days of culture was used to assess the activity. With pyrimethamine, proguanil, cycloguanil, primaquine, and 2 of its analogues (WR242511 and WR238605), marked inhibition of schizont formation could be achieved at concentrations below those causing a cytotoxic effect on the host hepatocytes. Chloroquine had only minimal schizonticidal activity at a concentration that produced severe hepatocyte toxicity. This simian in vitro system provides a reliable model for screening antimalarial compounds and for investigating their effects on the hepatic stage of malaria parasites.

Cultivation of exoerythrocytic stages of Plasmodium cynomolgi, P. knowlesi, P. coatneyi, and P. inui in Macaca mulatta hepatocytes.

Exoerythrocytic stage parasites of Plasmodium cynomolgi, P. knowlesi, P. coatneyi, and P. inui were cultured by inoculating primary cultures of hepatocytes from Macaca mulatta with sporozoites. Less than 1% of inoculated sporozoites survived. Morphology and size of the liver stages in all 4 species were similar to in vivo descriptions and the time required for in vitro maturation correlated well with prepatent periods described.