The American Journal of Tropical Medicine and Hygiene - Volume 46, Issue 1, 1992

Many of us have just returned from what is seen as one of the most successful meetings ever of the American Society of Tropical Medicine and Hygiene. Registered attendance was more than 1,200. Many of the meetings were standing room only. Symposia were pertinent, well-organized and heavily attended, and there were approximately 475 individual oral and poster presentations. Clearly the ultimate goal of these meetings—communication—was achieved in Boston. The membership of the Society owes a deep debt of gratitude to the local arrangements committee under the apt direction of John and Roberta David. Physical arrangements were remarkably free of problems. Coffee breaks, breakfasts, receptions and the banquet were enjoyable and gave a relaxed character to the meeting that should set standards for the future. Society & Association Services Corporation, the firm that handles the overall affairs of the ASTM&H, proved to be very effective in its role as meeting manager.

Malaria continues to be of enormous importance in various areas of the developing tropics. The first four papers in this issue are concerned with the subject of therapy for this disease. Two reports by Brasseur and colleagues address the problem of resistant strains of Plasmodium falciparum in Cameroon. It is interesting that the prevalence of chloroquine- and quinine-resistant isolates has remained essentially stable since the rapid emergence of this problem in 1985. The second of the two papers confirms the existence of mefloquine resistance in the country and correlates this phenomenon with the previous loss of quinine sensitivity in the same area. On page 15 is a report from Chinese colleagues of a new herbal agent, Daphnetin, with anti-malarial activity. The fourth paper in this issue presents the results of a clinical investigation on the impact of chloroquine prophylaxis during pregnancy on infant birthweight. In this study there was no significant difference in birthweights between treated and control groups.

The status of drug-resistant Plasmodium falciparum in Cameroon was determined in 1987–1988 in 221 children who were selected for chloroquine and quinine in vitro microtests. Resistance to quinine was found in 17% of 72 isolates from the southern part of the country, and in 7% of 87 isolates from the northern part of the country. The effective concentrations of 50% and 90% (EC50 and EC90) differed little from those observed in 1986. In southern Cameroon, 38 (51%) of 74 individuals harbored chloroquine-resistant isolates. The EC50 ranged from 8 to 553 nmol and the mean ± SD EC50 was 154 ± 148 nmol. In contrast, in the northern area, all but one of the 120 subjects studied had EC50 values below the cutoff limit of 80 nmol (mean = 20 nmol). In vivo 7-day assays performed with chloroquine at a dose of 25 mg/kg in 389 individuals from the southwestern part of the country clearly confirmed RII–RIII levels of resistance in 18–52% of the cases, depending on the location studied. In the northern area, in vivo 7-day assays at a chloroquine dose of 10 mg/kg showed 36 of 39 subjects studied to have drug-sensitive parasites.

Based on these results, it appears that after a rapid emergence of resistance that occurred in southern Cameroon, the prevalence of chloroquine- and quinine-resistant parasites remained fairly stable from 1986 to 1988. The stable difference between the northern and southern areas is believed to be related to both the lower rate of transmission and lower chloroquine drug pressure in the north.

To further document the phenomenon of Plasmodium falciparum resistance to mefloquine formerly described in Cameroon, complementary in vivo and in vitro studies were conducted. Two hundred six P. falciparum isolates were studied in vitro using an isotopic microassay with mefloquine solutions prepared daily. Using the cutoff limit of 30 nmol, 26 (20%) of 133 isolates from the northern part of the country were defined as being resistant to mefloquine. In contrast, only one of 73 isolates collected in the southern part of the country was resistant. In vivo 7-day assays were performed in the northern area in 57 asymptomatic P. falciparum carriers (age range 1–10 years) who were given a single 25 mg/kg dose of mefloquine (Lariam). Among 46 cases in which followup was possible, P. falciparum asexual parasites were cleared within five days in 38 cases, by days 6 and 7 in two cases, and remained detectable up to day 7 in six cases. Thus, these latter patients have a RII–RIII level of resistance by in vivo criteria. No resistance was found in 40 additional patients studied similarly in the southern region. These observations were made before any mefloquine drug pressure was exerted in the country, but results of cross-resistance and drug consumption studies support the hypothesis that in the northern region, where a close correlation (r = 0.67) was found between the response to quinine and mefloquine, the more frequent use of quinine may have induced a primary quinine resistance and a secondary mefloquine resistance (without chloroquine resistance). Chloroquine drug pressure in the southern region, which is 2.5 times higher than in the northern region, possibly led to a primary chloroquine resistance and a secondary quinine resistance (without mefloquine resistance).

Daphnetin is a dihydroxycoumarin that is being used in China for the treatment of coagulation disorders. It is also a chelator and an antioxidant. In vitro, daphnetin causes a 50% inhibition (IC50) of 3H-hypoxanthine incorporation by Plasmodium falciparum at concentrations between 25 and 40 µM. Several related compounds, such as scopoletin, 2, 3-dihydroxybenzoic acid and 3, 4-dihydroxybenzoic acid show no inhibitory activity. The antimalarial activity of daphnetin is inhibited by the addition of iron. Daphnetin does not appear to be an oxidant drug, since it does not spontaneously generate superoxide in vitro. However, it does alkylate bovine serum albumin when incubated in the presence of iron. In vivo, daphnetin significantly prolongs survival of P. yoelli—infected mice.

To determine the effect of chloroquine chemoprophylaxis during pregnancy on birth weights, a randomized trial was carried out in 1987 and 1988 in Banfora, Burkina Faso (West Africa). Seven hundred forty-five randomly selected women treated with chloroquine sulfate were compared to with 719 controls who received no treatment. In spite of an unquestionable effect of chloroquine in preventing placental infection (4.1% infected placentas in the treated group versus 19.0% in the controls), the mean difference in birth weights between the two groups (6 g) was not significant. The difference in the proportion of low birth weight (LBW) newborn babies in two groups (16.3% versus 16.4%) was also not significant. However, there was a strong relationship between placental infection and birth weight (the mean birth weight difference between infected and uninfected placentas was 113 g, and the proportion of LBW babies was 26.0% in infected placentas versus 14.8% in uninfected placentas). The small difference in birth weights observed between the two groups may be due to the fact that the prevalence rate of placental infection is low and that prophylaxis is effective only on a portion of the subjects in the treated group. It may also indicate that malaria is only one of several risk factors responsible for LBW. The relatively small increase in birth weight, the expected poor acceptance of mass prophylaxis, and the spreading of chloroquine-resistant Plasmodium strains should be considered before extending malaria chemoprophylaxis to all pregnant women. It might be worth considering to limit prophylaxis to primigravidae.

Anopheles mascarensis De Meillon, 1947, a mosquito that is native to Madagascar, is reported for the first time to act as a vector of Plasmodium falciparum malaria. From September 1989 to March 1990, 2, 499 An. mascarensis specimens from different regions of Madagascar were tested by an enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies against the circumsporozoite (CS) protein of the four human species of Plasmodium. The salivary glands of 237 specimens were also dissected. Fourteen of 1, 864 specimens obtained from Sainte Marie island on the Malagasy east coast were found by ELISA to be positive for the CS protein of P. falciparum. In addition, two of 237 specimens that were dissected were observed to have sporozoites in the salivary glands. These sporozoites were identified as P. falciparum by ELISA. In the other regions studied, no positive specimens were found. Due to observed behavioral differences between east coast and highland populations of An. mascarensis, the possible presence of a species complex is discussed.

A simple and highly sensitive assay for the detection of Trypanosoma cruzi antigens by spotting samples on nitrocellulose membrane filters is described. The immunobilized antigens are analyzed by subsequent binding of specific anti-T. cruzi immunoglobulins and secondary enzyme-labeled antibodies. Trypomastigote exoantigens were determined by this technique in the supernatant from cell cultures two days after infection. The T. cruzi circulating antigens present in serum specimens collected from mice infected with the parasite were demonstrable by day 3 of infection, when the parasitemia was still low. Antigenemia was also demonstrable by the dot-immunobinding assay in 14 of 16 congenitally infected patients. No cross-reactivity was observed using sera from healthy individuals, as well as from patients with other related parasitoses. The assay proved to be easy to perform and requires only small volumes of samples.

An enzyme-linked immunosorbent assay (ELISA) has been developed to detect antibodies in human sera to synthetic peptides derived from the repeating amino acid sequence in recombinant Trypanosoma cruzi antigens. Sixty serum samples from patients with chronic Chagas' disease were used to determine the reactivity against the synthetic repeat peptides derived from clones 1, 2, 30, 36, and shed acute phase antigen (SAPA). Ninety-eight percent of the samples had detectable antibodies to one or more of the synthetic peptides at titers > 1:100. The percentage of reactive sera increased from 28% with peptide SAPA to 93% with peptide 2. The exposure of patients to T. cruzi was reflected in indirect immunofluorescent antibody titers to fixed epimastigotes. Comparisons between ELISA and immunoradiometric assay data indicated that both tests were of approximately equal sensitivity.

A panel of eight Trypanosoma cruzi antigens produced by recombinant DNA techniques was used to compare the reactivity of IgG specificities in the sera from 45 chronic Chagas' disease patients with different clinical symptoms (cardiac disease, gastro-intestinal lesions, and combined syndrome) with those present in the sera from 55 asymptomatic patients in Chile. All of the serum samples were first characterized for antibody to T. cruzi epimastigotes by immunofluorescence assay. All of the Chagas' disease sera were reactive, but none of five healthy controls whose sera were also tested had antibodies against the fixed parasites. A dot-blot assay was then performed to evaluate the serum reactivity against recombinant DNA clones 1, 2, 13, 26, 30, 36, 54, and SAPA (shed acute phase antigen). These recombinant antigens were recognized by a large proportion of the sera collected from the Chilean patients. Ninety-five percent of the serum samples reacted with one or more of the recombinant clones. Analysis of the reactivity with individual fusion proteins showed that 88% of these sera reacted with clones 1 and 2, and 78% reacted with clone 13. Differences in reactivity to clones number 13, 30, and SAPA were observed when symptomatic and asymptomatic patients were compared. These differences in reactivity were statistically significant (P < 0.01) according to Fisher's exact test.

We have constructed a genomic DNA library of the Toxoplasma gondii ZS2 strain and isolated a specific cloned DNA sequence from this organism. The restriction map of this cloned 1.1-kb DNA fragment was analyzed. Southern and dot-blot analyses showed that the 32P-labeled DNA fragment hybridized to parasite DNA, to DNAs from peripheral blood leukocytes and the thymus of baby pigs that were artificially infected with T. gondii, and to DNAs of T. gondii-positive anencephalic and hydrocephalic fetuses. It did not hybridize with DNA from controls, (i.e., normal human and baby pig peripheral blood leukocytes, spleen of normal mice, Plasmodium falciparum, Pneumocystis carinii, and pBR322). As few as 100 T. gondii parasites or 500 pg of purified DNA from T. gondii can be detected by dot-blot hybridization. This probe method was specific and sensitive, and has been used successfully in detecting various clinical cases of toxoplasmosis with T. gondii.

During an epidemic of visceral leishmaniasis in the Sudan, two cases of congenital kala-azar were seen. The first child, whose mother had contracted kala-azar in southern Sudan, was born in Khartoum, where no transmission of leishmaniasis is currently occurring. At seven months, the child had fever, lymphadenopathy, and hepatosplenom-egaly; leishmania parasites were detected in the bone marrow. The child died and an autopsy showed leishmania parasites in all tissues including the lungs, kidneys, and thymus. In the second case, parasites were found in the placenta of a five-month-old fetus. These two cases demonstrate the importance of followup of infants born to mothers with leishmaniasis.

A cross-sectional survey of 348 subjects without evidence of liver disease was conducted to investigate the prevalence and risk factors for hepatitis C virus antibody (anti-HCV) seropositivity in the Yemen Arab Republic. The mean age of study subjects was 28.7 years (range 3–80), and 61% were males. Using commercial enzyme-linked immunosorbent assays (ELISA), 6.0% (95% confidence interval [CI] 3.8–9.1) of subjects were anti-HCV—positive, 13.5% were hepatitis B surface antigen-positive (HBsAg—positive), and 51.4% were positive for at least one serologic marker of prior hepatitis B infection. Nine (2.6%; 95% CI 1.2–4.9) of the 21 ELISA-positive sera were confirmed to be anti-HCV positive by a recombinant immunoblot assay. Anti-HCV seropositivity was significantly associated with age (odds ratio [OR] 2.0 for each 10-year increase in age) and prior surgery (OR 10.1), but was not associated with a history of prior blood transfusion or markers of hepatitis B infection. These preliminary data suggest that hepatitis C may pose a substantial health threat in Yemen.

Flocks of sentinel domestic pigeons (Columbia livia) detected increases in St. Louis encephalitis (SLE) and western equine encephalomyelitis (WEE) virus activity in southern California concurrently with flocks of sentinel chickens. However, occasional low-titered, transient seroconversions to both WEE and SLE viruses also occurred in pigeons during periods when virus activity was not detected by seroconversions in sentinel chickens, by virus isolation from Culex mosquitoes, or by human disease. Moreover, SLE virus seroconversions detected in pigeons by a hemagglutination inhibition (HI) test frequently could not be confirmed either by a plaque-reduction neutralization test (PRNT) on the same sera, or by an HI test on the next monthly serum sample from the same bird. Experimental infection studies, in which pigeons were inoculated subcutaneously with SLE (SOUE 16–84) virus, confirmed that pigeons developed low-titered and transient HI antibodies that were detectable infrequently by PRNT. In contrast, experimental infection with WEE (BFS 1703) virus produced elevated antibody responses that were detectable by HI for 8–12 weeks and by PRNT for at least 25 weeks. Pigeons infected with SLE virus rarely developed detectable viremias, whereas most birds infected with WEE virus developed viremias on postinfection day 1 that persisted for two or three days. Host-preference studies indicated that pigeons were less attractive as bait in lard can traps to host-seeking Culex mosquitoes than were chickens, and that blood-engorged Culex females collected near sentinel locations fed more frequently upon galliform than columbiform birds. Collectively, these results indicated that sentinel pigeons would not provide an adequate replacement for sentinel chickens to monitor WEE or SLE viruses, and would be a deadend host for SLE virus.

A two-site enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Gnathostoma spinigerum antigens in the sera of parasitized mice. This assay used IgG fractions prepared from serum of a G. spinigerum-infected rabbit as the capture antibody. The same IgG fractions were labeled with alkaline phosphatase and used as an antibody probe. The antigen detection assay was performed along with an antibody detection assay during the course of G. spinigerum infection in mice. Circulating antigen was detected after the first week of infection. The amount of detectable antigen increased steadily until the fourth week, but no significant amount of circulating antigen was detected thereafter. Serum antibody first appeared at the second week. Its level increased steadily until the fourth week, then remained high for a least eight weeks. The sensitivity of the two-site ELISA was approximately 6.75 ng/ml of larval somatic antigen and 27 ng/ml of excretory-secretory antigen. The assay gave false-positive results with Opisthorchis, Trichinella, and Angiostrongylus antigens at the level of 1, 728, 432, and 864 ng/ml or higher, respectively. This antigen detection assay may have application in the diagnosis of human gnathostomiasis.

A case of neurocysticercosis in a six-year-old Mexican boy and a case of Taenia solium taeniasis in his five-year-old brother are reported. Neurocysticercosis was suspected based on clinical findings and was confirmed by computed tomography scanning. A parasitologic examination with zinc-sulfate flotation and formalin-ether sedimentation techniques was carried out on the whole family, and revealed Taenia sp. eggs in three stool samples from the five-year-old boy. The entire family agreed to undergo chemotherapy with niclosamide, but only the child passing taeniid eggs eliminated T. solium. No additional taeniasis cases were found in an examination of 20% of the village population, using the same parasitologic techniques. The results of an ELISA using cysticercus antigens were negative for the boy with neurocysticercosis, for other family members, and for 24 village volunteers, but were positive for the T. solium tapeworm carrier. It was concluded that in this family, person-to-person transmission of the tapeworm occurred due to poor living conditions and hygiene.

We have identified specific ultrasonographic changes in Schistosoma japonicum—infected patients associated with serologic indicators of general liver function. An ultrasonographic examination concomitant with hematologic and biochemical serum analyses was performed on 102 patients at the Schistosomiasis Hospital in Leyte, The Philippines. The ultrasonographic liver images were classified into four patterns, according to the development of periportal fibrosis and the patterns of echogenic bands. Eleven cases with a long-term infection showed typical septal formation (network pattern). Other ultrasonographic changes in the portal system, such as the severity of splenomegaly, did not correlate with the age of the study patients or the duration of their infection; however, the production of collateral vessels was clear in the group of older patients. Among various hematologic and biochemical serum indicators of liver damage, the serum levels of total bile acid (TBA) and procollagen-III-peptide (P-III-P) strongly correlated with the development of hepatic fibrosis and portal hypertension. These findings suggest that the ultrasonographic liver patterns classified here, along with the changes in serum levels of TBA and P-III-P, provide useful indicators for field monitoring of S. japonicum infection.

We previously reported ultrasonographic and serologic abnormalities in 102 patients infected with Schistosoma japonicum in Leyte, The Philippines. These patients were subsequently treated with praziquantel (3 × 20 mg/kg), and changes in ultrasonographic images and the serum levels of liver function markers in 52 patients were followed up every three months for a period of 17 months. Improvement in the thickening of the portal vein wall and the intensity of echogenic bands was detected six months after treatment with praziquantel. The level of splenomegaly was also reduced in 42 patients who originally did not show the production of collateral vessels. A significant decrease in the serum total bile acid (TBA) level was detected in all patients six months after treatment with praziquantel. However, significant ultrasonographic changes could not be detected in the patients classified as type 3, with severe hepatic fibrosis caused by the long-term infection. These results clearly show that ultrasonographic examination, along with data on the serum TBA level, provides a sensitive tool to monitor the severity of hepatic fibrosis and portal hypertension caused by S. japonicum infection, as well as the improvement resulting from praziquantel treatment.