Antagonism of sulfadoxine and pyrimethamine antimalarial activity in vitro by p-aminobenzoic acid, p-aminobenzoylglutamic acid and folic acid.

The activity of pyrimethamine and sulfadoxine against two strains of Plasmodium falciparum has been studied in vitro by a radioisotopic technique. Low level antagonism of pyrimethamine resulted from the inclusion of p-aminobenzoic acid, p-aminobenzoylglutamic acid or folic acid in the test medium. Sulfadoxine activity was antagonised slightly by p-aminobenzoic but not by p-aminobenzoylglutamic acid, and antagonised markedly by folic acid at concentrations above 4 X 10(-8) M. At 10(-7) M folic acid, a concentration lower than that of normal RPMI medium 1640, sulfadoxine activity was reduced 7000 to 9000-fold in comparison with controls. These results are of importance in terms of the utilisation of folates by P. falciparum, the susceptibility of the parasite to antifolate drugs and the in vitro determination of parasite susceptibility.

Giardia lamblia and Cryptosporidium infections in child day-care centers in Fulton County, Georgia.

Risk factors for the introduction, spread and persistence of Cryptosporidium and Giardia lamblia infections in child day-care centers are not well understood. In 1989 and 1990 stool specimens were obtained from 292 diapered children attending 17 randomly selected day-care centers in Fulton County, GA; 8 (2.7%) children in 2 centers were infected with Cryptosporidium and 21 (7.2%) children in 7 centers were infected with Giardia. In 1986 the prevalence of Cryptosporidium and Giardia in these same centers had been 0.4 and 11.0%, respectively; the prevalence of Cryptosporidium, but not Giardia, increased significantly (P = 0.04) between 1986 and 1989 to 1990. Risk factors for Giardia infection included day-care attendance for greater than 3 months, the presence of toddlers in the classroom and the presence of other children in the household. Day-care centers with a Giardia-positive child in 1986 were not more likely to have an infected child in 1989 to 1990. Cryptosporidium, like Giardia, may be endemic in day-care centers in Fulton County.

Electrocardiographic changes during treatment of leishmaniasis with pentavalent antimony (sodium stibogluconate).

Serial electrocardiograms (ECGs) were obtained during 65 courses of sodium stibogluconate treatment in 59 Kenyan patients with leishmaniasis (56 visceral and 3 cutaneous). ECG abnormalities developed during 54% of the treatment courses. The frequency with which abnormalities occurred was related to the total daily dose of antimony (Sb), increasing from 2/9 patients treated with 10 mg Sb/kg/d to 25/48 treated with 20-30 mg Sb/kg/d and 8/8 treated with 40-60 mg Sb/kg/d. The frequency with which ECG abnormalities developed was also related to the duration of treatment, increasing from 11/65 patients after 7 days to 18/44 after 15 days, 26/39 after 30 days and 11/12 after 60 days. ECG abnormalities were similar to those previously described during treatment with trivalent antimonial drugs, the most common being flattening and/or inversion of T waves. Prolongation of the corrected QT interval occurred in 13 patients, all of whom were treated for more than 30 days or with more than 20 mg Sb/kg/d. One patient died suddenly during the fourth week of treatment with 60 mg Sb/kg/d, and 2 patients died of measles after 9 or 10 days of treatment with 30 mg Sb/kg/d. QT prolongation and a concave ST segment developed in all 3 patients who died. We conclude that minor ECG abnormalities are common when sodium stibogluconate is used at doses above 20 mg Sb/kg/d for more than 15 days, and that life-threatening arrhythmias may occur if very high doses are used.

Sensitivity of Plasmodium falciparum isolates to chloroquine in Kisumu and Malindi, Kenya.

The chloroquine sensitivity of Plasmodium falciparum isolates from infected persons living in Kisumu and Malindi, Kenya, was determined in vivo and vitro. There was no evidence of chloroquine resistance in 217 patients with P. faliparum infections who underwent standard W.H.O. 7-day in vivo tests. In 71 extended 35-day in vivo tests parasitemia recurred in 14 patients on days 21, 28, or 35. Parasites isolated from these 14 persons during the following period were tested in vitro. Eight tests were successful and showed the isolates to be chloroquine sensitive in vitro, suggesting that the recurrence of parasitemia resulted from reinfection rather than resistance. Macro in vitro tests were done on an additional 67 infected persons, 11 of whom also had sensitive 7-day in vivo tests. Chloroquine resistance was not demonstrated in vitro. In Malindi 100% of isolates were inhibited by a chloroquine concentration of less than or equal to 0.75 nmol/ml blood and 80% by less than or equal to 0.5 nmol as compared with 69% and 27.3% respectively of those from Kisumu. These data from individuals living in malarious areas of Kenya contrast with continuing reports of proven chloroquine-resistant P. falciparum malaria in non-immune visitors who acquired their infections in Kenya.

Chloroquine resistance in Plasmodium falciparum from Kenyan infants.

Forty-two infants, aged 6 to 24 months, infected with Plasmodium falciparum were identified in Kisumu, Kenya. Because of their age, all were presumably not semi-immune to malaria. Each infant was treated with 25 mg/kg chloroquine base and followed for 7 days. Forty-one infections were sensitive to chloroquine in vivo; asexual parasites disappeared in all by day 4 and were not present on days 5, 6, or 7. One infection was resistant in vivo; parasites disappeared by day 3 but recrudesced on day 4. Rieckmann micro in vitro tests for chloroquine were done on the 42 isolates. Interpretable results were found in 25. In vitro resistance was demonstrated in 18 (72%) isolates, including the patient with in vivo resistance; greater than or equal to 99% inhibition of schizont development only occurred in wells containing greater than or equal to 8 pmol chloroquine base (compared with less than or equal to 5.7 pmol/well for known sensitive isolates). This is the first demonstration of in vivo and in vitro chloroquine-resistant P. falciparum in a Kenyan. Comparison of these results with results from other studies carried out in the same area on the same area on older individuals suggests that the immune response may be playing a role in modifying the expression of resistance.

The enzyme-linked immunosorbent assay (ELISA) for malaria. I. The use of in vitro-cultured Plasmodium falciparum as antigen.

Using the Panama II strain of Plasmodium falciparum obtained from continuous in vitro culture as antigen, the micro enzyme-linked immunosorbent assay (ELISA) was used to test serum samples from 50 persons from the southeastern United States and serum specimens collected weekly from four non-immune and nine semi-immune patients infected with P. falciparum. None of the 50 sera from the United States had ELISA antibody titers greater than 1:80. The nine semi-immune patients had rapid ELISA antibody responses (titers greater than 1:2560) following patent parasitemia. ELISA titers remained elevated despite disappearance of patent parasitemia, and declined gradually following curative antimalarial therapy. The ELISA responses observed in the four non-immune patients were more variable, though positive titers appeared rapidly with patent parasitemia. Maximum titers were lower than those observed in semi-immune patients. These results demonstrate that P. falciparum obtained from continuous in vitro culture is an excellent antigen for the micro-ELISA test for malaria. However, further assessments of the ELISA are needed to identify the conditions associated with positive responses.

Evaluation of the micro enzyme-linked immunosorbent assay for antibodies to Trypanosoma cruzi.

A micro enzyme-linked immunosorbent assay (ELISA) for antibodies to Trypanosoma cruzi was evaluated and the results obtained by ELISA were compared with those obtained by the complement fixation test (CF) and indirect fluorescent antibody test (IFA). Fifty sera collected from residents of the southeastern United States all had reciprocal ELISA titers less than or equal to 320. Similarly, serum samples from 17 patients with T. cruzi infection proven by xenodiagnosis had reciprocal ELISA titers of greater than or equal to 1,280. Specimens from 302 El Salvador Army recruits were tested by ELISA, IFA, and CF. Excellent correlation was observed between results obtained by the three serologic tests; 62.9% of the samples were negative by each of the three tests and 24.5% were positive by all. Overall, 29.5% of the sera were positive for antibodies to T. cruzi by ELISA, 29.5% by IFA, and 31.5% by CF. The data suggest that the micro ELISA is a promising serologic test for measuring antibodies to T. cruzi in individuals and in populations.

Evaluation of UNICEF/Arab Republic of Egypt/WHO schistosomiasis Control Project in Beheira Governorate.

We evaluated the UNICEF/Government of Egypt/WHO Schistosomiasis Control project in 2 districts of Beheira Governorate of the Nile Delta during 3 weeks in February 1988. The project, begun in 1983, was focused on reducing prevalence, intensity, and morbidity due to schistosomiasis by providing diagnosis and treatment with praziquantel to schoolchildren. Schools were visited twice. Following the completion of the school surveys, the program was extended into the community. Chemotherapy was delivered by mobile and static teams. The evaluation indicated that, with respect to accuracy of diagnosis, record-keeping, and coverage of targeted populations, project tasks were performed exceedingly well by highly motivated, well-supervised mobile teams. Static teams in rural health centers were less successful in providing diagnosis and chemotherapy to village populations. We resurveyed 6 randomly selected schools to assess the impact of chemotherapy. Overall, the prevalence of Schistosoma mansoni infection was reduced from 60.3% to 24.8% between the first and second surveys (approximately 1 year apart) and was still lower (41.1%) than initial levels up to 3 years after the last treatment with praziquantel. The percentages of those with greater than or equal to 34 S. mansoni eggs/slide using the Kato-Katz technique showed a marked and prolonged decrease (17.1% to 0.3% to 2.2%). The prevalence of S. haematobium infection dropped from 37.6% to 5.5% and was still 9.9% at the time of the evaluation. The percentages of those with greater than or equal to 50 S. haematobium eggs/10 ml urine dropped less dramatically (17% to 4.4% to 11.9%). Mobile teams conducting vigorous chemotherapy programs targeted at schoolchildren can have long-lasting benefits in terms of prevalence and intensity.

Intestinal parasitism in the United States: update on a continuing problem.

To document patterns of intestinal parasitism in the United States, we analyzed results of 216,275 stool specimens examined by the state diagnostic laboratories in 1987; parasites were found in 20.0%. Percentages were highest for protozoans: Giardia lamblia (7.2%), Entamoeba coli and Endolimax nana (4.2% each), Blastocystis hominis (2.6%), and Entamoeba histolytica (0.9%). The most commonly identified helminths were nematodes: hookworm (1.5%), Trichuris trichiura (1.2%), and Ascaris lumbricoides (0.8%). Identifications of G. lamblia increased broadly from the 4.0% average found in 1979, with 40 states reporting increases and seven reporting decreases. Seasonally, Giardia identifications increased in the summer and fall, especially in the Midwest. Nine states reported hookworms in more than 2% of specimens; none were states with indigenous transmission. We analyzed similar, but abbreviated, data for 1991; parasites were found in 19.7% of the 178,786 specimens and Giardia was found in 5.6%. States reporting percentages of Giardia identification in the highest quartile for both 1987 and 1991 were located in the Midwest or in the Northwest. Cryptosporidium was identified in both the 1987 and 1991 surveys; it had not been identified in a previous survey. For each year, Cryptosporidium was reported from 25 states across the country (for both years in 17 states). We conclude that intestinal parasitism should not be overlooked as a cause of gastrointestinal illness in the United States and that the prevalence of Giardia may be increasing.

Endemic amebiasis in an extended family.

Two cousins from a large Spanish-American family were simultaneously diagnosed as having amebic liver abscesses. Survey of 183 extended-family members revealed that 45.7% of 162 had a positive amebiasis indirect hemagglutination test and 12.6% of 111 had cysts or trophozoites of Entamoeba histolytica demonstrated in a single stool examination. A total of five family members had had liver abscesses; two deaths had occurred. In a random sample survey of the remainder of the community, only one person (0.3%) had a positive serologic test. Within the extended family, person-to-person appeared to be the predominant mode of transmission. Water supplies were not contaminated. Both community and extended family homes had the same source of water. Type and source of food supply were not correlated with infection and there was no evidence to implicate an infected food handler. Clustering of seropositivity occurred in homes without indoor toilets. Homes of the extended family were more crowded and significantly fewer of them had indoor toilets. Endemic foci of amebiasis continue to exist in the United States. Follow-up family and other close contacts of persons with amebiasis will frequently identify other cases.

In vitro antimalarial activity of tetrahydrofolate dehydrogenase inhibitors.

Three tetrahydrofolate dehydrogenase (dihydrofolate reductase = EC 1.5.1.3) inhibitors were tested for antimalarial activity against Plasmodium falciparum, using an in vitro radioisotopic technique. Activity of each drug was tested in both normal RPMI medium 1640 and in modified medium (containing no p-aminobenzoic acid and 2.27 X 10(-8) M folic acid) after a 24- or 48-hour exposure. Activity was increased 20- to 85-fold using the modified medium and the longer exposure time. Under all conditions, pyrimethamine and cycloguanil were of equal or greater potency than an experimental pyrimethamine analogue, M&B 35769, against pyrimethamine-sensitive strains, but M&B 35769 was more active than either pyrimethamine or cycloguanil against pyrimethamine-resistant strains.

Field evaluation in Kenya of a 48-hour in vitro test for Plasmodium falciparum sensitivity to chloroquine.

A 48-hour in vitro test for determining the chloroquine sensitivity of Plasmodium falciparum isolates was evaluated in Kisumu and Malindi, Kenya. P. falciparum isolates from 14 children, aged 5 to 13 years, were studied. In vivo and 48-hour in vitro tests were done on all 14. Successful Rieckmann macro and micro in vitro tests for chloroquine sensitivity were completed in nine isolates each. All 14 infections cleared within 3 days of beginning chloroquine treatment, and none recrudesced during a 7-day (8 patients) or 28-day (6 patients) follow-up period. The three in vitro tests gave comparable results. Although all isolates tested were chloroquine sensitive in vitro, different response patterns were observed. In the 48-hour test, 10 isolates were inhibited at chloroquine concentration less than or equal to 0.03 nmol/ml medium. These isolates were inhibited by less than or equal to 0.5 nmol of chloroquine per ml blood in the Rieckmann macro test and by 2-6 pmol/well in the micro test. The other four isolates had response patterns intermediate between those of previously reported sensitive and resistant strains. Complete inhibition did not occur until chloroquine concentrations of greater than or equal to 0.03 nmol/ml medium in the 48-hour test, greater than or equal to 0.5 nmol/ml blood in the macro test, and 6 pmol/well in the micro test. The results demonstrate that the 48-hour test is a useful addition to existing in vivo and in vitro methods for determining the chloroquine sensitivity of P. falciparum in the field.

The Duffy blood group and resistance to Plasmodium vivax in Honduras.

To test the hypothesis that the Duffy blood group negative genotype is a factor in resistance to Plasmodium vivax, we determined the Duffy blood group, the malaria antibodies, and the slide-demonstrated infection rates with P. vivax and P. falciparum of 420 persons living in Nueva Armenia, Honduras. In all, 247 persons were Duffy negative. Demonstrated infections with P. falciparum were almost equally distributed between Duffy-positive (5,8%) and Duffy-negative (4.9%) persons. Similarly, Duffy-positive (25.6%) and Duffy-negative (28.2%) persons had equal proportions of indirect fluorescent antibody test titers suggestive of past or present P. falciparum infection. In contrast, all 14 P. vivax infections were found in Duffy-negative persons. There was no evidence suggesting that Duffy-positive and Duffy-negative persons had different exposures to malaria. The Duffy negative genotype FyFy appears to be a factor in resistance to P. vivax.

Diarrhea in a non-hospitalized rural Salvadoran population: the role of enterotoxigenic Escherichia coli and rotavirus.

To determine the role of rotavirus, enterotoxigenic Escherichia coli and enteropathogenic E. coli in diarrheal disease of non-hospitalized children and adults living in rural El Salvador, stool specimens were collected from 156 persons with diarrhea and 134 age- and sex-matched controls over a 1-year period. Enterotoxigenic Escherichia coli (ETEC) were isolated as frequently from controls (13.4%) as from diarrhea cases (12.2%). Enteropathogenic E. coli were isolated from 13 cases (8.3%) and 10 (7.7%) controls. Rotavirus was demonstrated in only five of the 129 specimens from cases examined; the five persons infected were less than or equal to 3 years of age. No invasive E. coli were found. Serotyping of ETEC revealed serogroups of ETEC previously associated with enterotoxigenicity but was not helpful in separating infection from disease. The etiology of diarrhea in this rural, non-hospitalized population was complex. Isolation of a known pathogen did not prove etiology. The rotaviruses, which have been isolated frequently from hospitalized persons, were rare. Further laboratory and epidemiologic studies in such populations are needed to identify those factors that determine pathogenicity.

Recent trends in the prevalence and distribution of schistosomiasis in the Nile delta region.

In 1983, a survey of 71 villages in the Nile delta demonstrated that the overall prevalence of Schistosoma mansoni and S. haematobium infections was 39% and 5%, respectively. Recent increased availability of praziquantel, combined with Egyptian Ministry of Health-sponsored media efforts to educate the public about schistosomiasis, prompted us to determine the current status of S. mansoni and S. haematobium infections in the delta and evaluate any changes that may have occurred since the previous survey. The same villages that participated in the 1983 survey were resampled in 1990. Stool and urine samples were requested from all occupants over the age of two years in a 5% sample of houses within each village. Stool (Kato) thick smears and urine sediments were read qualitatively at the rural health station. Field-prepared Kato smears and a 20% sample of urine specimens were forwarded to the Ministry of Health Laboratory, where quantitative readings were also performed. Analysis of samples obtained from 17,310 persons revealed that S. mansoni prevalence had decreased to 23% and that S. haematobium prevalence had decreased to 3% (P < 0.001). The highest levels of schistosome infection were found in governates located in the eastern section of the delta. The observed changes in the prevalence of S. mansoni and S. haematobium suggest that control measures are having a favorable impact on schistosomiasis transmission in this region.

Alterations in filarial antigen-specific immunologic reactivity following treatment with ivermectin and diethylcarbamazine.

The presence of circulating microfilariae has been associated with alterations in B and T cell functions. In this study, we compared the influence of diethylcarbamazine (DEC) and ivermectin on filarial antigen-specific immune responses in a Haitian population. Both drugs were effective at reducing microfilaremia levels to less than 10% of pretreatment levels for up to one year. This reduction in microfilaremia was associated with two phases of altered cellular responsiveness monitored with in vitro assays. Five days post-treatment, cellular proliferation in response to both filarial and nonfilarial antigens was significantly increased, as was the background response in the absence of any antigen. At both nine months and one year post-treatment, the filarial antigen-specific reactivity of both DEC- and ivermectin-treated patients was significantly increased over baseline levels. No differences were observed between the two treatment groups in terms of humoral or cellular reactivity to filarial antigens, despite evidence suggesting a role for DEC in adult worm killing. These results provide additional evidence that microfilariae modulate antifilarial immune reactivity.

Antibody response to heterologous and homologous antigens in Brugia malayi- and B. pahangi-infected Mongolian jirds as measured by the enzyme-linked immunosorbent assay (ELISA).

Sequentially collected sera from Mongolian jirds (Meriones unguiculatus) infected with Brugia malayi and B. pahangi were tested for antibodies to homologous and heterologous antigens by the enzyme-linked immunosorbent assay (ELISA). Titers were less than 1:100 prior to infection and rose rapidly (within 2 weeks). Peak titers were observed prior to patent microfilaremia, and high titers persisted during infection. Use of the homologous antigen did not increase sensitivity or specificity of the ELISA. In fact, B. malayi-infected jirds demonstrated higher titers to the heterologous antigen, B. pahangi, than to the homologous antigen. Fractionation of B. malayi antigen over a wide pH range using isoelectric focusing did not eliminate cross-reactions, but the reactions of 20 B. malayi sera and 20 B. pahangi sera tested more strongly to specific fractions, particularly in the lower pH range.

Serologic and parasitologic studies of Entamoeba histolytica in El Salvador, 1974-1978.

Serologic and parasitologic studies were done in El Salvador, C.A., from 1974-1978 to examine the reliability of the diagnosis of Entamoeba histolytica infection in an endemic area and to confirm the estimates of morbidity and mortality due to amebiasis. The results suggest that infection with E. histolytica is common, but the estimated prevalence is too high. Misdiagnosis occurring in hospital and public health laboratories was documented. Data collected from examining family members of persons with E. histolytica infection and disease indicate that most infections are asymptomatic, and support the hypothesis that estimates of morbidity and mortality rates are excessive.

The enzyme-linked immunosorbent assay (ELISA) for malaria. III. Antibody response in documented Plasmodium falciparum infections.

Serum specimens from patients in El Salvador, Central America, with slide-proven Plasmodium falciparum infections were examined for antibodies to P. falciparum using the enzyme-linked immunosorbent assay (ELISA) and the indirect fluorescent antibody (IFA) methods. Both serologic tests were positive in 78.1% of the 827 samples, both negative in 5.4%, the ELISA positive alone in 6.3%, and the IFA alone in 10.2%. Agreement between the serologic tests was better in the specimens with high positive titers (high IFA = high ELISA). Seropositivity rates and geometric mean titers were higher in the older (greater than or equal to 15 years) age groups for both ELISA and IFA; in such persons, the IFA was positive in 92% and the ELISA in 88%. The lowest seropositivity rates found by the ELISA were observed in children; 27.6% of 98 children less than or equal to 4 years of age were negative. A longer duration of infection as evidenced by the presence of gametocytes on the blood slide resulted in higher positivity rates by both ELISA and IFA. This phenomenon, particularly apparent in young children, supports the belief that the more important variable in determining the proportion of false negatives is previous malaria experience and not age. The results indicate that, while neither serologic test is appropriate as a diagnostic aid, both the ELISA and the IFA would be useful in epidemiologic investigations.

Comparative efficacy of clearing-dose and single high-dose ivermectin and diethylcarbamazine against Wuchereria bancrofti microfilaremia.

To compare the efficacy and tolerability of various combinations of low- and high-dose ivermectin and diethylcarbamazine (DEC), 59 persons with Wuchereria bancrofti microfilaremia were enrolled in a double-blinded six-arm clinical trial in Leogane, Haiti. On day 1, study participants were treated with low clearing doses of ivermectin, DEC, or placebo; on day 5 they received 200-400 micrograms/kg of ivermectin or 6 mg/kg of DEC. Adverse reactions, which were generally mild, occurred more frequently with ivermectin than with DEC. One year after treatment, the geometric mean microfilarial density returned to 0.9% of pretreatment levels for persons who received a total of 420 micrograms/kg of ivermectin. This rate was significantly lower than 5.6% for persons who were treated with 220 micrograms/kg of ivermectin (P = 0.02) and 9.3% for those receiving 6 or 7 mg/kg of DEC (P = 0.006). Persons treated with a clearing dose of ivermectin followed by 6 mg/kg of DEC also had low microfilarial densities (1.7% of pretreatment levels), suggesting an additive or synergistic effect of the two drugs. The addition of a clearing dose neither reduced the severity of adverse reactions nor improved the efficacy of high-dose ivermectin. Community-based intervention trials are now warranted to determine the feasibility and effectiveness of mass chemotherapy with single high-dose ivermectin for the prevention and control of lymphatic filariasis.