The American Journal of Tropical Medicine and Hygiene - Volume 56, Issue 3, 1997

We conducted a three-year entomologic study in Dielmo, a village of 250 inhabitants in a holoendemic area for malaria in Senegal. Anophelines were captured on human bait and by pyrethrum spray collections. The mosquitoes belonging to the Anopheles gambiae complex were identified using the polymerase chain reaction. Malaria vectors captured were An. funestus, An. arabiensis, and An. gambiae. Anopheles funestus was the most abundant mosquito captured the first year, An. arabiensis in the following years. The annual entomologic inoculation rates calculated by enzyme-linked immunosorbent assay were 238, 89, and 150 for the first, second, and third years, respectively. Each year there was a peak of transmission at the end of the rainy season, but transmission occurred year round. The heterogeneity of transmission was found at four different levels: 1) the relative vector proportion according to the place and method of capture, 2) the human biting rate and relative proportion of vectors by month and year, 3) the infection rate of each vector by year, and 4) the number of infected bites for all vectors, and for each species, for the year. Our data show that even in areas of intense and perennial transmission, there exist large longitudinal variations and strong heterogeneity in entomologic parameters of malaria transmission. It is important to take these into account for the study of the variations in clinical and biological parameters of human malaria, and to evaluate this relationship, a very thorough investigation of transmission is necessary.

Using an indirect immunofluorescence assay on stool samples, we found a 6.4% prevalence of cryptosporidiosis among 403 children less than five years of age with acute diarrhea in Mexico City over a one-year period. The prevalence was highest (11.4%) during the rainy summer months. Most Cryptosporidium parvum cases occurred in infants less than one year of age. Cryptosporidium parvum was more common in malnourished children (P < 0.05) and in nonbreast-fed infants less than six months of age (P < 0.01). Neither dwelling characteristics nor the presence of domestic animals at home were associated with C. parvum infection. Enteropathogenic bacteria were found in 26.8% of the children; Escherichia coli, Salmonella, and Shigella being the most frequently identified. None of 100 serum samples tested showed antibodies against human immunodeficiency virus. When compared with immunofluorescence, the acid-fast technique showed a sensitivity of 76.9% and a specificity of 98.9%, with a predictive positive value of 83.3%. It was concluded that 1) monoclonal antibody-based immunofluorescence is a better diagnostic tool than the acid-fast technique, 2) the prevalence of cryptosporidiosis in this population was similar to that of other developing countries, 3) clinical manifestations were nonspecific, and 4) C. parvum was more common in malnourished children and in nonbreast-fed infants.

We followed 1,756 young, rural Bangladeshi children less than five years of age for one month after identification of sentinel Shigella patients in their neighborhoods. Two hundred nineteen (12%) children developed Shigella diarrhea (shigellosis) and 227 (13%) developed culture-negative dysentery. Shigella flexneri (60%) and S. dysenteriae, type 1 (15%) were the most common isolates among shigellosis cases. Within individual neighborhoods, there was poor agreement (Kappa = 0.21) between Shigella species isolated from sentinel patients and from additional cases detected during surveillance. The risk of shigellosis increased substantially after infancy and peaked in the second year of life. Severe stunting, as assessed by height-for-age, was associated with an increased risk of shigellosis (adjusted odds ratio [ORa] = 1.67, 95% confidence interval [CI] = 1.09–2.57, P < 0.05), while breast-feeding was protectively associated (ORa = 0.40, 95% CI = 0.24–0.69, P < 0.001). Only 43% of the shigellosis cases reported bloody stools; frank dysentery occurred more frequently in S. dysenteriae 1 infections than in S. flexneri infections (ORa = 5.04, 95% CI = 1.76–14.48, P < 0.01), and was also associated with severe stunting (ORa = 2.16, 95% CI = 1.01–4.58, P < 0.05). Our findings show that the high risk of shigellosis in residentially exposed Bangladeshi children results from multiple Shigella strains circulating concurrently within the same neighborhood; demonstrate that the risk is notably modified by host age, nutritional status, and dietary patterns; and illustrate that the classic picture of dysentery is relatively infrequent and is correlated with the infecting species and with host nutritional status.

In two areas of Senegal where previous evidence of Rift Valley fever (RVF) virus circulation was detected, Barkedji in the Sahelian bioclimatic zone and Kedougou in the Sudano-Guinean zone, a longitudinal study of the enzootic maintenance of RVF virus was undertaken from 1991 to 1993. Mosquitoes, sand flies, and ticks were collected and domestic ungulates were monitored with serologic surveys. Rift Valley fever virus was not isolated in Kedougou. In Barkedji, RVF virus was isolated from Aedes vexans and Ae. ochraceus mosquitoes collected in traps near ground pools and cattle droves and from one healthy sheep. Sand flies were not involved in the maintenance cycle. Seroconversions were recorded in three (1.9%) of 160 monitored sheep and goats. The interepizootic vectors appeared to belong to the Aedes subgenus Neomelaniconion in East Africa, and to the subgenus Aedimorphus in West Africa. Epizootics in East Africa are associated with an increase in rainfall. However, factors associated with epizootics remain unknown for West Africa.

The distribution and prevalence of antibody reactive with Sin Nombre virus were determined in mammals in biotic communities of the southwestern United States. Small mammals (n = 3,069) of 69 species were trapped in nine communities from lower Sonoran desert to alpine tundra. Antibody was found in rodents from all communities (overall prevalence = 6.3%); prevalence was lowest at the altitudinal and climatic extremes (0.4% in desert and 2.0% in alpine tundra). Antibody occurred in 11% of 928 deer mice, 20% of 355 brush mice, 23% of 35 western harvest mice, and 12% of 24 Mexican voles. No infected deer mice were found in desert habitat; prevalence varied from 4% in chaparral to 17% in pinyon-juniper. Brush mice were frequently infected in chaparral and montane forest (25%). Seropositivity was higher in males and in heavier animals, suggesting horizontal transmission among adult males. Decreasing prevalence with age among the youngest deer mice suggests that infected dams confer passive immunity to pups.

Women and their infants may benefit from therapeutic interventions when hepatitis B, human immunodeficiency virus (HIV), or syphilis are detected during the prenatal period. We initiated hepatitis B and HIV screening of women attending prenatal clinics in Belize. Risk factor assessment information for hepatitis B infection and demographic data were determined by interview. Of 543 evaluable women, 81 (14.9%) were seropositive for hepatitis B core antibody (anti-HBc); one woman had asymptomatic hepatitis B surface antigenemia. Antibodies to HIV-1 were detected in one woman. Reactive syphilis serologies were detected in 15 (2.8%) women. Anti-HBc seroprevalence varied by district (range 3.1–43.5%) and by ethnicity (range 0.0–40.9%). Significant identified risks for anti-HBc seropositivity from univariate analyses included being of the Garifuna ethnic group, residence or birth in the Stann Creek or Toledo districts, a reactive syphilis serology, a household size of eight or greater, and five or more lifetime sexual partners. Multivariate analyses identified ethnicity and a reactive rapid plasma reagin as the best predictors of anti-HBc seropositivity. Highly variable differences in anti-HBc prevalence by district may permit the targeting of limited public health resources for education, screening, and prevention programs.

During the past decade, effective snake antivenoms have become scarce in northern Nigeria. As a result, many patients severely envenomed by the saw-scaled or carpet viper (Echis ocellatus), which is responsible for more than 95% of the snake bites in the region, did not receive effective treatment and mortality and morbidity increased. To combat this crisis, a new monospecific ovine Fab antivenom (EchiTab™) is being developed. Its theoretical advantages over conventional equine F(ab′)2 antivenom are a more rapid tissue penetration and larger apparent volume of distribution (the volume of [tissue] fluid in which the the antivenom would be uniformly distributed to achieve the observed plasma concentration). In a preliminary study, two vials (20 ml; 1.0 g of protein) of EchiTab rapidly and permanently restored blood coagulability and cleared venom antigenemia in seven envenomed patients. Four experienced early reactions that responded to epinephrine. In a randomized comparative trial of one vial (10 ml; 0.5 g protein) of EchiTab or four ampules (40 ml; 2.12 g of protein) of Institute Pasteur Serum (Ipser) Africa polyspecific F(ab′)2 antivenom, there were fewer reactions, but only 36% and 35% of patients, respectively, showed permanent restoration of coagulability, with the remainder requiring further doses. This suggests that 0.5 g (one vial) of EchiTab is approximately equivalent to 2.12 g (four ampules) of Ipser Africa antivenom, and that a higher initial dose will be required for most patients. Measurements of circulating venom and antivenom levels reflected the clinical events.

Blastocystis hominis is a common intestinal parasite of humans in the tropics whose pathogenic role is in dispute. Its presence has been reported in a variety of intestinal disorders resembling irritable bowel syndrome (IBS) such as diarrhea, anorexia, and flatulence. We have therefore investigated a possible link between IBS and blastocystosis by determining IgG antibody levels to B. hominis in patients with IBS. Levels of IgG antibodies were significantly elevated in patients with IBS compared with asymptomatic controls (P < 0.0001, by Student's t-test) in both B. hominis stool culture-positive and stool culture-negative IBS patients. When IgG antibodies were divided into their respective subclasses, only IgG2 levels were significantly increased in IBS patients compared with asymptomatic controls, indicating that the predominant response in these patients may be directed to carbohydrate antigens. The diagnostic usefulness of this test in IBS patients remains to be established because these data are only suggestive of a possible link between B. hominis and IBS. However, we hope that this antibody test will help in elucidating the controversy that surrounds the role of B. hominis as a pathogen at present.

We conducted a retrospective analysis of 43 consecutive children (35 boys and 8 girls), 4–14 years of age and living in an urban area, who were hospitalized at the Instituto de Infectologia Emilio Ribas (Sao Paulo, Brazil) from January 1989 to December 1995 with an acute illness subsequently diagnosed as leptospirosis. Epidemiologic data indicated contact with contaminated water in most cases (88%). The patient sera reacted most strongly with Leptospira interrogans serovars copenhageni (45%) and icterohaemorrhagiae (32.7%). Jaundice was present in 70% of the patients, elevated transaminase levels in 56%, renal failure in 79%, meningitis in 23%, thrombocytopenia in 65%, and hemorrhagic manifestations in 11.6%. Three children had pulmonary hemorrhage with respiratory failure and one death occurred as a consequence of respiratory failure. We also observed that antimicrobial therapy reduced the extent of renal failure and thrombocytopenia. These data indicate that antibiotics benefit children with late, severe leptospirosis and that severe disease also occurs in children and should be considered in the differential diagnosis.

A community study on opisthorchiasis was conducted in Prachinburi Province in eastern Thailand during 1990–1992. The morbidity from opisthorchiasis in the community and reversibility of biliary pathology following treatment with praziquantel at a single dose of 40 mg/kg were assessed by longitudinal investigations of clinical, laboratory, and ultrasonographic changes. A total of 913 voluntary subjects infected with Opisthorchis viverrini were randomly selected for longitudinal study, and 579 subjects without liver fluke infection were recruited as controls. The majority of the study group suffered from mild and moderate infections that were associated with nonspecific gastrointestinal symptoms. Grade I and II ultrasonographic changes, which indicated chronic inflammation of the biliary tract and gallbladder, were detected in 32% of the infected individuals. Clinical symptoms and ultrasonographic changes were common in subjects 21–40 years of age and older. Satisfactory resolution of morbidity was observed during two years follow-up on days 0, 60, 180, 360, and 720, as shown by significant clinical improvement, normalization of laboratory parameters, and downgrading of ultrasonographic abnormalities. Portable ultrasonography has proved to be a reliable noninvasive technique in the evaluation of the morbidity due to opisthorchiasis in rural areas.

Atovaquone (dihydroxynaphthoquinone 566C80) is a broad-spectrum antiprotozoal compound demonstrating potent antimalarial activity against multidrug-resistant malaria. We present the results of in vitro drug sensitivity tests of 142 Plasmodium falciparum isolates, 108 from 14 countries of West and Central Africa, 32 from the Philippines, and one each from Laos and Myanmar. These were tested in vitro against nine drugs: the classic antimalarials chloroquine, quinine, mefloquine and halofantrine, the four qinghaosu derivatives, artemisinin, artemether, artesunate, and arteether, and the new compound atovaquone. Results showed the Asian strains have a higher median 50% inhibitory concentration (IC50) to almost all drugs compared with those from Africa. This was significantly different for chloroquine, halofantrine, and artemisinin. We used three different approaches to estimate the threshold for resistance of atovaquone to be approximately 5–7 nmol/L. The global median of 96 pooled strains is 1.4 nmol/L and the 90th percentile is 5.5 nmol/L for atovaquone. There were no correlations of atovaquone with the eight other antimalarials among African strains, but significant correlations, except for halofantrine, were observed among Asian strains. The absence of a correlation between atovaquone and the other available drugs indicates the potential of atovaquone as an alternative antimalarial in Africa. The correlation observed among Asian strains, however, suggests that atovaquone has to be used cautiously in Asia. Nevertheless, the association with proguanil in recently concluded clinical trials in Europe, South America, Asia, and Africa has demonstrated its antimalarial efficacy.

During an outbreak of dengue hemorrhagic fever (DHF) in Jakarta, Indonesia in 1988, we attempted to isolate dengue virus using mosquito cells and a medium containing heparin. Whole blood, immediately after being drawn from patients, was inoculated into Aedes albopictus cell cultures temporarily maintained in the heparin-containing medium. The overall virus isolation rate was 25% (17 of 69) samples collected within three days after admission of the patients to hospital. No virus was obtained thereafter. The successful virus isolation was apparently not related to titers of anti-dengue virus hemagglutination-inhibiting antibodies present in patients' sera. The viruses were recovered from cases of each of the four World Health Organization grades of DHF without significant differences. The technique is simple and easily performed at bedside.

An integrated system for sample preparation and DNA detection of the malaria parasite using immunomagnetic separation in combination with the polymerase chain reaction (PCR) and colorimetric analysis is described. A cocktail of three monoclonal antibodies towards merozoite surface antigen-1 was used for magnetic capture of parasites from microliter amounts of whole blood. A sensitivity down to a parasitemia of 10-6% was achieved using cultured parasites as a model. The integrated approach was evaluated in a field study. A total of 410 blood samples from patients attending malaria clinics in Trat province and Kanchanaburi province in Thailand were analyzed. The samples were processed by immunomagnetic separation and transferred to central laboratory for PCR-based detection. Microscopic examinations on blood smears were done in parallel; 53% were positive using the DNA-based assay, while only 32% were positive using conventional microscopic analysis. This field study suggests a possible model for large-scale testing of malaria with an increased sensitivity compared with conventional methods.

The release of reactive oxygen intermediates (ROI), mediators of inflammatory reactions, was evaluated in murine Trypanosoma cruzi infection. In acutely infected BALB/c mice, spleen cells were stimulated, either with epimastigote or trypomastigote forms of the parasite, and the effect was enhanced by serum from infected mice. Only opsonized parasites triggered the release of ROI by normal mouse cells and this response was several times lower than in infected mice. This seems to indicate that cells from acutely infected mice reacted to T. cruzi and that neither parasites nor serum factors blocked the release of ROI. During the acute stage of the infection, both the parasitemia and the release of ROI by spleen cells were higher in BALB/c than in C3H mice (ROI generated in response to a phagocytic stimulation was 12 and 3 times the normal levels, respectively). In addition, in BALB/c mice infected with different numbers of parasites, the production of ROI was related to parasitemia. On the other hand, during the chronic stage of the infection, the inflammatory reaction in myocardium was greater in C3H than in BALB/c mice, and the increase in ROI production was 30% and 100% above the normal levels in BALB/c and C3H mice, respectively. This suggests that the increased ROI production paralleled the parasite burden in the acute phase, and could be related to inflammatory processes after the control of the parasitemia.

Hyperbaric oxygen (HBO) has been reported by some to be therapeutic for necrotic lesions induced by the venom of the brown recluse spider, Loxosceles reclusa. Others have reported no efficacy for this treatment. In this study, the effect of high pressure oxygen on an enzymatic activity of the toxin of this venom is reported. The time course for the hydrolysis of the phosphocholine ester bond of chicken egg yolk sphingomyelin, as catalyzed by brown recluse spider venom (BRSV) and venom treated with extended HBO (12 hr at 10 atmospheres), was followed by phosphorus-31 nuclear magnetic resonance spectroscopy. The venom and HBO-pretreated venom demonstrated sphingomyelinase D activity. Phospholipase C activity was not detected. The sphingomyelinase D activity of BRSV in three separate experiments was not altered by HBO. The HBO-pretreated venom, in all cases, did not exhibit an altered time course in the overall hydrolysis of the D linkage of sphingomyelin.

Polymerase chain reaction (PCR) analysis is a widely used method for detection of Borrelia burgdorferi DNA in biological specimens, including ticks. Studies have demonstrated that substances present in mammalian blood can inhibit PCR amplification. This would limit the utility of PCR for determination of B. burgdorferi infection in engorged ticks that have taken a blood meal from a human or other animal host. To systematically assess the potential for such inhibition, nymphal Ixodes scapularis, which had acquired B. burgdorferi as larvae, were fed on rats. These engorged ticks were lysed in standard PCR lysis buffer and aliquots were subjected to PCR analysis; 0 of 56 were PCR positive. An equivalent cohort of unfed (unengorged) ticks had an infection rate of 19% (11 of 57) as determined by identical PCR analysis (P = 0.0006, by Fisher's exact test). When lysates from the engorged ticks were spiked with the 500 prelysed B. burgdorferi, none of the samples yielded a positive PCR signal, indicating the presence of inhibitory substances. Consistent with this observation, PCR amplification of the original engorged tick lysates after extraction with a DNA extraction kit, resulted in detection of B. burgdorferi DNA in 13 specimens (23%). Furthermore, when 500 prelysed B. burgdorferi were added to the treated extracts, all samples (56 of 56) were PCR positive. Thus, extraction resulted in removal of inhibitors of PCR amplification present in unprocessed engorged tick lysates. Furthermore, additional titration experiments showed that some inhibitory substances may also be present in unfed ticks, although this inhibition does not completely prevent detection of B. burgdorferi DNA in unprocessed lysates. This study clearly demonstrates that inhibitors of PCR amplification are present in engorged ticks and prevent accurate determination of B. burgdorferi infection rates by this method unless steps are taken to remove such inhibitors.

We have previously shown that Plasmodium falciparum recombinant antigens PfEB200, R23, and Pfi72 consistently inhibit opsonization of infected red blood cells by protective hyperimmune Saimiri sera, indicating that they present target epitopes involved in the phagocytosis of infected red blood cells. We report here an analysis of the immune response elicited in naive squirrel monkeys injected with the individual recombinant antigens or with a mixture of the three antigens combined with a synthetic peptide. In the three administration protocols investigated, there was no evidence for the production of antibody contributing to the phagocytosis of infected red blood cells, contrasting with the increase of opsonizing antibodies elicited by these antigens in monkeys with a prior (≥ 500 days) experience with malaria infection. However, the recombinant antigens were highly immunogenic, inducing specific antibody responses to P. falciparum and to the recombinant antigens. When the monkeys immunized with the antigen combination were challenged with blood-stage parasites, there was substantial protection: three of seven immunized animals self-cured and two others experienced a delayed peak of parasitemia. Taken together with our previous findings, these results suggest that PfEB200, R23, and Pfi72 constitute interesting vaccine candidates, and show that the presence of antibodies promoting phagocytosis of infected red blood cells is not a prerequisite for protection after immunization with these antigens in the Saimiri model.

The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify selected lymphokine mRNAs from phytohemagglutinin-activated leukocytes of the owl monkey (Aotus trivirgatus). Interleukin-2 (IL-2), IL-4, IL-13, and interferon-gamma were selected as lymphokine mRNAs of interest, since expression of these cytokines helps define the type of T helper lymphocyte response (i.e., TH1 versus TH2). Because sequences for these lymphokine genes were not available for the owl monkey, multiple PCR primers for each lymphokine gene were designed based on published human sequences. Various PCR primer pairs were then used in the RT-PCR to determine the conditions for optimal amplification of each owl monkey cytokine mRNA. In addition, each PCR primer pair was compared for the ability to amplify lymphokine mRNAs from other primate species, including African green (Cercopithecus aethiops), squirrel (Saimiri sciureus), and rhesus (Macaca mulatta) monkeys. The specificity and sensitivity of optimal primer pairs was also demonstrated by amplification of as little as 10 fg of each lymphokine gene in a background of 300 ng of irrelevant cDNA. Finally, partial sequences of owl monkey coding regions for IL-2, IL-13, and interferon-gamma were determined and compared for homology with their human counterparts. Together, these studies define specific and sensitive conditions for detection of lymphokine mRNA expression in the owl monkey and provide partial sequence information of the coding region for these lymphokines. This investigation should provide molecular probes to investigate the immune response against malaria and the effectiveness of malaria vaccines in the owl monkey that models this human disease.