The American Journal of Tropical Medicine and Hygiene - Volume 54, Issue 4, 1996

In a study group of 183 people in a Schistosoma mansoni-endemic area in Burundi, stool examinations were performed with duplicate 25-mg Kato-Katz slides on seven occasions (days 1, 3, 5, 8, 10, 32, and 37). Point prevalences detected by single examinations of 25 mg and 50 mg of stool varied from 41.0% to 57.9% and from 55.7% to 63.9%, respectively. The cumulative prevalence for all seven measurements was 82.0%. The individual day-to-day variation in egg output was important. The majority of infections missed by the examination of single slides and specimens were light ones. The Kato-Katz method applied on a single stool specimen is more suitable for morbidity control, but less suitable for control of infection. When a precise quantitative diagnosis on the individual level is required, several measurements on different days are necessary. The data presented validate recently developed statistical models and charts predicting true prevalences.

We have used the nested polymerase chain reaction (PCR) to assay for low level Plasmodium falciparum infections that were below the threshold of detection of blood film examination. This revealed a substantial group of asymptomatic, submicroscopically patent infections within the population of a Sudanese village present throughout the year although clinical malaria episodes were almost entirely confined to the transmission season. In our September, January, April, and June surveys, the PCR-detected prevalences were 13%, 19%, 24%, and 19%, respectively. These figures reveal a much higher prevalence of dry season infection than previous microscopic surveys have indicated. Furthermore, 20% of a cohort of 79 individuals were healthy throughout the September to November transmission season but were PCR-positive for P. falciparum in a least one of a series of samples taken in the ensuing months. Levels of exposure to P. falciparum infection were therefore higher than was previously believed in this region, highlighting the fact that many individuals were infected but healthy for most of the year. The reservoir parasite population was thus larger and more stable than previously thought, a finding that is consistent with the high levels of genetic variation at polymorphic loci reported from analysis of P. falciparum parasites in this area.

On the Cherokee Indian Reservation and surrounding area of western North Carolina, an area-wide serosurvey was conducted to determine the prevalence of neutralizing antibody to La Crosse (LAC) virus. A questionnaire was used to identify risk factors important in exposure to virus-infected mosquitoes in populations near the reservation. Of 1,008 serum samples tested, 9.6% were positive for LAC virus antibody. For samples solely collected from on (n = 311) or off (n = 697) the reservation, the prevalence of seropositive samples was 20.6% on the reservation and only 4.7% off the reservation. Seropositivity increased directly with age, indicating that transmission of LAC virus was highly endemic. Age and location residence (on versus off the reservation) were significant risk factors for exposure to LAC virus. Persons on the reservation were 5.5 times more likely to have been exposed to LAC virus than were people who reside off the reservation. An additive increase in risk of 1.5 times over each age group was found, so that the oldest age group (≥ 75 years) was 7.5 times more likely to have been exposed to LAC virus than was the youngest age group (< 1–14 years).

To further understand the role of wild mammals in the maintenance of La Crosse virus (LACV) in nature, we investigated the effects of inoculation method and virus source on the duration and amplitude of LACV viremia in vertebrate hosts. Earlier work suggested that deer are not sufficiently susceptible to LACV to play an important role in its maintenance. We re-evaluated the susceptibility of deer since subsequent studies showed that they constitute 65% of Aedes triseriatus blood meals, and thus would be exposed frequently to the virus. In our study, deer developed higher and longer viremias following exposure to LACV by infected Ae. triseriatus than those previously reported by inoculation with needle and syringe. However, susceptible Ae. triseriatus that fed on these viremic animals did not become infected. Because a large number of uninfected mosquitoes can feed upon a viremic deer in nature, we believe that deer should not be disregarded completely as a possible amplifier in the LACV transmission cycle. We also infected chipmunks to determine if there were significant differences in viremia response from mosquito delivery of virus to the chipmunk host, compared with artificial exposure by injection. Chipmunks exposed to infected mosquitoes had higher and longer viremias than the ones produced by intramuscular injection of an LACV suspension. These findings show the importance of using LACV infected mosquitoes for transmission experiments in mammals.

This study analyzed histopathologic specimens of 600 pediatric solid malignant tumors seen during the period 1979–1994 at the histopathology laboratories of the Rift Valley Provincial General Hospital in Nakuru, the Nyanza Provincial General Hospital in Kisumu, and the Uasin Gishu Hospital in Eldoret in western Kenya. The crude incidence rate of each malignancy per 100,000 children per year was calculated. The patterns of malignancies were examined with a focus on tumor incidence, age, sex, geographic, and ethnic distribution to relate the tumors to putative environmental and genetic causative factors. The six common tumors were Burkitt's lymphoma (33.5%), non-Hodgkin's lymphoma (21.8%), retinoblastoma (11.5%), Kaposi's sarcoma (6.1%), nephroblastoma (4.5%), and Hodgkin's disease (4.1%). Significantly high crude incidence rates for lymphomas and Kaposi's sarcoma showed a characteristic ethnogeographic distribution. The majority of the tumors were found concentrated around Lake Victoria and showed decreasing occurrence as one moved towards the semi-arid and highland areas. We concluded that environmental factors seem to play a major role in childhood tumors in western Kenya.

We studied the fluctuations of schistosome circulating antigens in urine as compared with fecal egg counts in 60 Burundese individuals infected with Schistosoma mansoni. Levels of circulating anodic antigen (CAA) and circulating cathodic antigen (CCA) in the urine were determined by quantitative enzyme-linked immunosorbent assays. Fecal samples were simultaneously collected and examined with duplicate Kato-Katz slides. Significant correlations were consistently found between circulating antigen levels in urine and fecal egg counts. Although both antigen levels and egg output fluctuated, there was less fluctuation of CCA levels in urine than of fecal egg counts. All individuals had CCA in at least one urine sample and 82% were at least once positive for egg counts. Positive CAA levels were found in at least one urine sample in 75% of all individuals, but levels were low. Our results show that detection of CCA in urine is a sensitive, quantitative, and reliable method for noninvasive diagnosis and screening of S. mansoni infections, due to the relatively low fluctuations.

A microplate-type enzyme-linked immunosorbent assay for the detection of Taenia species antigen in human feces was tested in field studies undertaken in two Guatemalan communities. The test was based on immunoglobulin G antibodies from a rabbit hyperimmunized to Taenia solium proglottides. Comparison was made with microscopy and patient interviews as a means of diagnosis. The coproantigen test result was positive in 79 of the 1,582 fecal samples examined. Parasitologic confirmation was made in 55 of these cases. The coproantigen test was the most sensitive technique used, detecting 2.6 times as many confirmed cases of taeniasis as microscopy, which diagnosed 21 cases. Only one case was detected by interviewing. Microscopy revealed one false-negative coproantigen result. Mass treatment of the population did not result in the detection of any additional cases. Twelve coproantigen-positive results were categorized as unconfirmed and an additional 12 as putative false-positive results, giving an overall specificity of 99.2% for the coproantigen test. Of the 34 taeniid tapeworms identified to the species level, all were T. solium. The practicalities of the use of such a test in epidemiologic studies on human taeniasis are discussed.

To identify Wuchereria bancrofti DNA sequences that could be used as the basis for a simple and rapid parasite detection assay, a genomic library of W. bancrofti was constructed and screened for highly repeated DNA. The repeat found with the highest copy number was 195 basepairs (bps) long, 77% AT, and 300 copies per haploid genome. This sequence was designated the Ssp I repeat because it has a unique recognition site for that restriction endonuclease in all or most of the repeat copies. The Ssp I repeat DNA family is dispersed, genus-specific, and exists in all of the different geographic isolates of W. bancrofti tested. Based on DNA sequence analysis of this repeat, we have developed an assay to detect very small quantities of W. bancrofti DNA using the polymerase chain reaction (PCR). With this PCR assay, the Ssp I repeat was detected in as little as 1 pg of W. bancrofti genomic DNA (about 1% of the DNA in one microfilaria) added to 100 µl of human blood. The PCR assay also amplified Ssp I repeat DNA from geographic isolates of W. bancrofti from around the world but not from other species of filariae or from human or mosquito DNA. Microfilaria-positive human blood samples collected in Mauke, Cook Islands were shown to be Ssp I PCR-positive, while microfilaria-negative samples were PCR-negative. The specificity and sensitivity of the Ssp I PCR assay indicates that this approach has significant potential for improved screening of large human populations for active W. bancrofti infection.

We have developed a highly sensitive method for detection of Francisella tularensis in clinical samples based on a nested polymerase chain reaction (PCR) for the FopA gene. Mice infected with F. tularensis were killed at 24-hr intervals, and the DNA from blood and spleens was extracted by a variety of methods and analyzed by PCR. The best method, based on the ability of DNA to bind to silica in the presence of guanidine thiocyanate, yielded amplifiable DNA without dilution of the murine tissues samples. Francisella tularensis in infected murine spleens and culture-positive blood samples was reliably detected by nested PCR following this extraction procedure. We believe this technique has significant advantages over traditional methods for diagnosing F. tularensis infection in terms of speed, ease of use, reproducibility, and safety.

An enzyme-linked immunosorbent assay (ELISA) was developed to detect different hantavirus antigens in cell culture; i.e. Puumala (PUU), Hantaan (HTN), and Dobrava (DOB) viruses. The assay was based on binding human serum immunoglobulin M (IgM) antibodies to the solid phase by use of goat anti-IgM antibodies. The captured IgM antibodies were present in the acute phase serum from two patients: one infected in Sweden and the other in Bosnia. Antigens being bound to the solid phase by the human anti-PUU and anti-DOB/HTN IgM antibodies were detected by a broadly reacting polyclonal rabbit anti PUU-recombinant nucleocapsid protein antiserum. The IgM isotype was proven to be at least five times more efficient than IgG when used as the capturing antibody. The sensitivity of the PUU antigen ELISA was approximately 0.5 ng/ml, as measured by titration with a PUU recombinant nucleoprotein antigen. Cell-associated PUU antigen in tissue culture was seen after 48 hr by the PUU-ELISA and after 96 hr by immunofluorescent assay. When tested for capacity to discriminate between PUU, DOB, and HTN viruses, significant differences were found: the Swedish serum detected PUU antigen at high titers, whereas no reactivity was found against DOB and HTN; the Bosnian serum detected both DOB and HTN at high titers but had a low reactivity to PUU. The method was also tested for its usefulness in detecting PUU antigen in bank vole (Clethrionomys glareolus) lungs. Of 59 animals captured from the surroundings of patients with nephropathia epidemica, three became positive with a high activity in the PUU-ELISA, but with low reactivity in the DOB/HTN-ELISA. It is concluded that a sensitive ELISA has been developed to detect different hantaviruses in cell culture and lungs of bank voles.

The Santa Lucia strain of Plasmodium falciparum and the Aotus lemurinus griseimembra monkey are proposed as models for the testing of sporozoite vaccines and transmission-blocking vaccines. Approximately 85% of splenectomized monkeys were infected when fed upon by 10 or more heavily infected Anopheles freeborni mosquitoes. Sporozoite-induced infections in monkeys with or without previous infection with P. vivax readily infected mosquitoes, thus making them candidates for testing transmission-blocking vaccines.

The Santa Lucia strain of Plasmodium falciparum and the Aotus vociferans monkey were studied as models for the testing of transmission-blocking vaccines. Virulence developed early in the passage history. Despite the use of only small quantities of chlorguanide and/or quinine to control infection coupled with the use of small inocula and delays in splenectomy, mosquito infection was markedly reduced from that seen during primary passage to this species of Aotus. It appears that the model may be most useful during its initial passage from the primary species, Aotus lemurinus griseimembra.

Products generated by filarial nematodes depress vascular reactivity by mechanisms involving endothelial cells. We hypothesized that comparable filarial-induced alterations might occur in lymph vessels. Experiments were designed to test the hypothesis that spontaneous contractions of bovine mesenteric lymphatics studied in vitro are altered by the filarial parasite Brugia pahangi. Rings of bovine mesenteric lymphatics were suspended in tissue baths and spontaneous contractions were evaluated for rate, rhythm, and amplitude. Rings that met inclusion criteria (rate > 1.8/min, regular rhythm, and an amplitude > 500 mg) were randomly exposed to B. pahangi or used as controls. Parasites were added to the bath at time zero. Changes in rate, rhythm, and magnitude of spontaneous contractions were evaluated every 10 min. Comparisons were made within control or Brugia-infected groups over time and between groups (B. pahangi versus controls). The presence of B. pahangi significantly depressed the frequency of spontaneous contractions when compared with controls. Control rings were stable over time, without changes in rate, rhythm, or amplitude. However, B. pahangi altered both the rate and rhythm of spontaneous contractions. Since spontaneous contractile activity is likely to be important in the propulsion of lymph, alterations of contractile activity could result in lymphedema. Thus, filarial factors may be responsible, in part, for altered lymphatic function seen in lymphedema. Pharmacologic intervention aimed toward influencing host-parasite metabolic interactions may, in this complicated scenario, prove useful.

The pig is a vital link in the transmission cycle of Taenia solium, the cestode responsible for human-porcine cysticercosis. Infected pigs also represent an important source of economic loss to farmers in developing countries. Past efforts to find an adequate therapeutic regimen to treat this parasitic disease in swine have failed because of low efficacy, high cost, side effects, or the need for multiple doses. In this randomized, no treatment-controlled study, the efficacy and safety of oxfendazole and praziquantel for the treatment of porcine cysticercosis were evaluated in 16 naturally infected pigs. Four groups of four pigs were treated with oxfendazole, praziquantel, oxfendazole plus praziquantel, or untreated. The pigs were humanely killed 12 weeks post-treatment, the number of cysts was counted, and parasite viability was assessed by cyst evagination. No detectable side effects were seen in any of the pigs. Praziquantel treatment alone appeared to reduce the number of cysts, but did not decrease the viability of the remaining parasites. Treatment with oxfendazole alone or oxfendazole plus praziquantel killed all of the parasites, and left only microcalcifications in the meat. Oxfendazole provides, for the first time, a practical, effective, inexpensive, and single-dose therapy for porcine cysticercosis.

Genomic DNA probes were made for two recently identified members of the Anopheles punctulatus complex; Anopheles sp. near punctulatus from Papua New Guinea and Anopheles farauti No. 7 from the Solomon Islands. The probes are species-specific and with the use of 32P labeling sensitive enough so that a squash blot of only a small segment of the mosquito is required for identification. The 119-basepair (bp) probe for An. sp. near punctulatus and the 1,106-bp probe for An. farauti No. 7 have been sequenced in full and the probes have been tested on field collected specimens. These probes now make it possible to distinguish An. sp. near punctulatus and An. farauti No. 7 from the other eight members of the An. punctulatus complex. A pan-species probe was also made from the 18S ribosomal DNA that binds to DNA from all members of the complex. These three probes complete the set required for distinguishing all known members of the An. punctulatus complex by DNA hybridization.

Oliveros virus is an agent isolated in cell culture from Bolomys obscurus (Rodentia, Muridae, Sigmodontinae) captured on the central Argentine pampa. Oliveros virus was shown to be related to members of the Tacaribe complex of the family Arenaviridae by immunofluorescent antibody (IFA) tests, electrophoretic pattern of viral proteins, and morphology as observed by electron microscopy. It was distinct from 12 other arenaviruses by a combination of plaque-reduction neutralization tests, comparison of endpoint titers among cross-IFA tests, and comparison of viral RNA sequence data. This agent is the third new arenavirus from South America described within the last three years.

Ehrlichia chaffeensis, an obligately intracellular bacterium with tropism for monocytes, is the etiologic agent of human monocytic ehrlichiosis. To determine the nature and ultrastructural location of E. chaffeensis antigens, monoclonal antibodies (MAbs) to E. chaffeensis were developed. The MAbs were used for immunofluorescence and Western immunoblotting analysis of the antigens of density gradient-purified ehrlichiae. Monoclonal antibody 6A1 recognized an epitope of a 30-kD protein. This antibody reacted with a strain-specific epitope of E. chaffeensis, Arkansas strain, and did not cross-react with any other ehrlichia tested. Monoclonal antibodies 3C7 and 7C1-B recognized Arkansas strain proteins of 30 and 29 kD and reacted with E. chaffeensis (strain 91HE17) proteins of 31 and 29 kD and an E. canis protein of 30 kD. Lack of reactivity of these two MAbs with E. sennetsu and E. risticii suggests that the epitope is group-specific. Monoclonal antibody 5D11 recognized a 58-kD protein of both strains of E. chaffeensis as well as E. canis, apparently a group-specific, conformation-independent epitope. Monoclonal antibody 7C1-C reacted with 58- and 88-kD proteins of both the Arkansas and 91HE17 strains. Trypsin treatment destroyed the reactivity of E. chaffeensis antigens with all the MAbs when tested by Western immunoblotting, indicating that these antigens are proteins with trypsin-sensitive epitopes. Immunoelectron microscopy of negatively stained intact E. chaffeensis organisms showed that the 30- and 29-kD proteins are present on the surface of the ehrlichial cell wall along with the previously localized 28-kD protein.

Forty-nine cases of murine typhus were diagnosed in recent years in residents of several communities around the city of Chalkis, the capital of the Prefecture of Evia. (Euboea) Evia is an island connected to central mainland Greece by a bridge. To investigate the endemicity of murine typhus in this area, 226 fleas (Xenopsylla cheopis) and blood samples were collected from 53 rats (Rattus norvegicus) trapped in this area. The polymerase chain reaction followed by restriction fragment length polymorphism analysis (PCR-RFLP) was used to detect and identify Rickettsia typhi, the etiologic agent murine typhus, in the rat blood samples (buffy coat cells) as well as in their fleas. An indirect immunofluorescent antibody (IFA) assay was performed to detect antibodies against R. typhi in rat serum samples. The presence of R. typhi in both fleas and rat blood samples was demonstrated. The frequency of infection for X. cheopis was 4%, while 18% of the rats had buffy coat cells infected, and 92% of the rat sera tested by IFA were positive for anti-R. typhi antibodies. The present work is the first successful application of PCR-RFLP in a field study of naturally infected rats and their fleas in Europe.

Thirty six stocks of Trypanosoma cruzi isolated from sylvatic mammals (32 Didelphis marsupialis and one Philander opossum) and triatomine bugs (Rhodnius robustus and one unidentified bug) in the Amazonian forest of Carajas, Brazil were characterized by isoenzyme and random amplified polymorphic DNA (RAPD) analysis as belonging to principal zymodeme 1 (Z1). Two different homozygous phenotypes and the corresponding heterozygous phenotype were found for phosphoglucomutase with an observed frequency almost identical with that predicted by the theoretical Hardy-Weinberg distribution. Parental and hybrid profiles were also suggested by RAPD analysis, which allowed exclusion of mixed parental strains from the hybrids: isoenzyme and RAPD profiles of biological clones were also indistinguishable from those of uncloned stocks. Trypanosoma cruzi stocks from widely separated geographic origins in Central and South America gave similar RAPD profiles that allowed them to be recognized as being Z1. These results indicate that genetic exchange could contribute to the generation of genetic diversity during the sylvatic cycle of T. cruzi, and this may have epidemiologic and taxonomic implications.

Karyotype analysis of 69 strains of Leishmania belonging to three species of the Viannia subgenus originating from the southeastern and southwestern regions of Colombia revealed approximately 5.3-kb RNAs in four strains of L. braziliensis and also in the World Health Organization reference strain L. guyanensis IWHI/BR/78/M5313. The RNA element in this reference strain and in L. braziliensis strains isolated from cutaneous and mucosal lesions of four patients hybridized with RNA probes prepared from cDNA of the RNA virus present in L. guyanensis strain CUMC-1-1A (LRV1-1). These strains also contained an 80-kD protein that reacted with polyclonal antibody prepared against a recombinant fragment of the coat (capsid) protein of LRV1-1. In addition, another Colombian strain of L. braziliensis was found to contain an approximately 3.5-kb RNA that did not hybridize with LRV1-1 probes. Contrasting with the strains containing the 5.3-kb RNA, a total lysate of this strain did not contain material reactive with antiserum to the capsid protein fragment. All Leishmania containing LRV1-related viruses identified to date have originated in the Amazon River basin. Karyotype analyses and biological characterization of 17 clones obtained from the highly metastatic L. guyanensis strain 5313 revealed retention of the approximately 5.3 kb RNA in all clones and no segregation of the virus with the metastatic trait. The restricted distribution of LRV1-related viruses among some strains of L. braziliensis and L. guyanensis circulating in the Amazon River basin makes these elements potential epidemiologic markers.

We used sequences specific to the small subunit ribosomal RNA (SSU rRNA) of the sporogonic stages of Plasmodium falciparum to design a reverse transcriptase-polymerase chain reaction (RT-PCR) assay that can detect 0.1 sporozoites in total RNA purified from potentially infected mosquitoes. We made a synthetic RNA that is amplified in the RT-PCR by the same primers as the parasite SSU rRNA and that serves as an internal control and competitive quantitation standard. We calibrated the assay for quantitation of sporozoites by making a standard curve with RNA from purified and counted sporozoites. The assay accurately measured sporozoite number with a linear range of at least three orders of magnitude in a single reaction. Some applications and limitations of the assay are discussed.