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- Volume 46, Issue 5, May 1992
The American Journal of Tropical Medicine and Hygiene - Volume 46, Issue 5, May 1992
Volume 46, Issue 5, May 1992
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In this Issue
Pages: iii–ivMore LessPathology and pathogenesis are subjects of high interest to a number of investigators. Plasmodium falciparum is the focus of the first two papers in this section which are from the Medical Research Council field station in Gambia, involving investigators from Sweden and the United Kingdom. In the first report (page 503), fresh isolates of P. falciparum from Gambian children were studied for their ability to spontaneously form erythrocytic rosettes. All isolates from children with cerebral malaria formed rosettes, but the phenomenon was observed in less than 60% of those from patients with uncomplicated disease. The second paper (page 511) addresses a physical characteristic associated with the phenomenon of cytoadherence in P. falciparum, the presence of knob-like protrusions on the surface of infected erythrocytes. The authors conclude that both knobbed and knobless phenotypes are found in natural infections with the parasite and that P. falciparum-infected erythrocytes are able to bind to a large number of human cell types.
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Rosette Formation in Plasmodium falciparum Isolates and Anti-Rosette Activity of Sera from Gambians with Cerebral or Uncomplicated Malaria
Pages: 503–510More LessAbstractThe ability of Plasmodium falciparum-infected red blood cells (RBC) to form spontaneous erythrocyte rosettes was studied in 130 fresh isolates from Gambian children with cerebral or uncomplicated malaria from August to November 1990. All isolates (24 of 24) from patients with cerebral malaria formed rosettes, but only 61 of 106 isolates from children with uncomplicated malaria formed rosettes. The mean rate of rosette formation in isolates from children with cerebral malaria (28.3%) was significantly greater than that in isolates from children with uncomplicated malaria (8.5%). Giant rosettes were more frequently formed in isolates from patients with cerebral malaria than in those from patients with uncomplicated malaria. Sera of children with cerebral disease generally lacked anti-rosette activity, while many sera from children with uncomplicated malaria showed strong anti-rosette activity when tested against the patients' own parasites. Some sera that were devoid of autologous rosette-disrupting activity were able to disrupt rosettes formed in other isolates, indicating the presence of different rosette formation mechanisms. Forty percent (6 of 15) of the sera from patients with cerebral malaria caused microagglutination of the patients' own uninfected and infected RBC, while only 10% (3 of 31) of sera from children with uncomplicated disease caused microagglutination. The ability of infected RBC to bind to melanoma cells grown in vitro did not differ between patients with cerebral or uncomplicated malaria. The results of this study, taken in conjunction with our previous findings, establish a strong association between rosette formation in P. falciparum-infected RBC and cerebral malaria.
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Ultrastructural Analysis of Fresh Plasmodium falciparum-Infected Erythrocytes and Their Cytoadherence to Human Leukocytes
Pages: 511–519More LessAbstractSixty fresh Plasmodium falciparum isolates obtained from Gambian children with mild or cerebral malaria were investigated by transmission electron microscopy for the expression of knob-like protrusions (K+) on the surface of the infected erythrocytes. More than six-hundred infected erythrocytes were analyzed. Knob-forming parasites were present in all 60 isolates. Although knobless parasites (K-) were found in 25 (42%) of the isolates, only 39 were K-, while 577 were K+. Nine of the 39 K- infected erythrocytes that were studied in greater detail appeared to be asexual parasites because they were either segmented or they lacked mitochondrial DNA-like filaments and cristae, which are abundant in immature gametocytes. No difference was observed in the relative frequency of K+K- infected erythrocytes in isolates from patients with mild or cerebral malaria. Binding of both knobby and knobless infected erythrocytes to autologous leukocytes including monocytes, neutrophils, lymphocytes and plasma cells was found in some of the primary in vitro cultures. By using P. falciparum laboratory strains of known phenotypes and leukocytes from healthy blood bank donors, it was established that this novel adherence phenomenon was related to that of cytoadherence to certain melanoma or endothelial cells. Cytoadherent infected erythrocytes that bind to leukocytes enhance antibody-independent phagocytosis and induce cellular aggregation, while non-cytoadherent or rosetting infected erythrocytes do not. We conclude that P. falciparum parasites of both the knobby and knobless phenotypes are present in natural infections, and that P. falciparum-infected erythrocytes are able to bind to a large number of human cell types. The direct binding of infected erythrocytes to immunocompetent cells may play a role in the immune response against the parasite.
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Release of Prostaglandin E2 by Microfilariae of Wuchereria bancrofti and Brugia malayi
Pages: 520–523More LessAbstractTo elucidate the local release of immunomodulatory prostaglandins by intravascular filarial parasites, the formation of prostaglandin E2 (PGE2) was examined in individual microfilariae of Wuchereria bancrofti and Brugia malayi. Following incubation of living microfilariae immobilized in an agar matrix, prostaglandins released by the parasites were fixed by carbodiimide and localized by indirect immunofluorescence. Prostaglandin E2 was specifically detected around the entire surface of microfilariae with anti-PGE2 antiserum, but not with control nonimmune or PGE2 affinity-immunoadsorbed antiserum. These results provide direct evidence that individual microfilariae of W. bancrofti as well as B. malayi release prostaglandins into their microenvironment. The release of PGE2 by these intravascular parasites may modulate host leukocyte responses, and thereby contribute to the immune defects observed in infected humans with peripheral microfilaremia.
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Genomic and Biologic Analyses of Snowshoe Hare Virus Field and Laboratory Strains
Pages: 524–532More LessAbstractLow-passage field strains of snowshoe hare (SSH) virus (Bunyaviridae), the prototype SSH virus (originally isolated in Montana), and La Crosse (LAC) virus were compared serologically by plaque-reduction neutralization (PRNT) and molecularly by oligonucleotide fingerprinting (ONF). The PRNT and ONF results confirmed the identity of the field strains, although some differences in the fingerprints were observed. We have examined the RNA genome variability in the two field and three laboratory strains of SSH virus, using direct sequence analysis of selected RNase T1 oligonucleotides. Few changes were observed among three Montana prototype-derived laboratory isolates, although they have different passage histories. In contrast, the field isolates differed greatly from the laboratory strains. In addition, we have located several of the larger T1 oligonucleotides within the known sequence of the small and large RNA genome segments. We then compared the viruses for their ability to replicate in and be transmitted by Aedes triseriatus mosquitoes. The oral infection rates for LAC, the field isolates, and the SSH prototype, as determined by immunofluorescent examination of midgut tissues, were 100%, 82%, and 47%, respectively. All viruses were also transmissible from mosquitoes to mice.
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Localization of a 230-KD Parasitophorous Vacuole Membrane Antigen of Plasmodium berghei Exoerythrocytic Schizonts (LSA-2) by Immunoelectron and Confocal Laser Scanning Microscopy
Pages: 533–537More LessAbstractUsing antiserum to a 230-kD parasitophorous vacuole membrane (PVM) antigen of Plasmodium berghei exoerythrocytic schizonts as a specific probe for the PVM, we studied the three-dimensional structure of this membrane within infected host cells by immunoelectron microscopy and confocal laser scanning microscopy at 3, 4, and 50 hr after sporozoite invasion. Fluorescent label was not detected at 3 hr, but was associated with the cytoplasm of 24-hr-old exoerythrocytic parasites. Specific labeling of the PVM was not observed by immunoelectron microscopy until 50 hr, when numerous vesicles and finger-like projections of the PVM were found in the cytoplasm of infected host cells. Labeled vesicles were often isolated and located at the periphery of the infected hepatocyte. Confocal microscopy demonstrated that these vesicles formed discontinuous chains that extended from 3–10 µm away from the parasite. These structures appear to be similar to the membranous clefts of Plasmodium-infected erythrocytes, and may be important in the movement of host or parasite proteins within infected hepatocytes.
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Occurrence of Severe Leptospirosis in a Breeding Colony of Squirrel Monkeys
Pages: 538–545More LessAbstractAlthough experimental leptospirosis has been studied in various species of monkeys, the occurrence of acute leptospirosis in a population of nonhuman primates is uncommon. We report on a number of severe cases of icterohemorrhagic leptospirosis that appeared in the squirrel monkey (Saimiri sciureus) colony of 109 animals at the Institute Pasteur in French Guiana. Initially, 11 animals had acute illness, with jaundice and a hemorrhagic syndrome, leading to 10 deaths. Two Leptospira interrogans strains were isolated from blood cultures of sick monkeys, and one was isolated from the urine of a rat trapped in the breeding park. All three belonged to serovar copenhageni, and tests using monoclonal antibodies showed that these three strains were extremely similar. In the following weeks, five pregnant female monkeys had miscarriages; two of them had antibodies against the Icterohaemorrhagiae serogroup. An epidemiologic study conducted on the 93 remaining animals demonstrated a seropositivity rate of 26% (microagglutination test [MAT] titer ≥ 100) primarily for the Icterohaemorrhagiae serogroup, but also for the Ballum, Grippotyphosa, Sejroe, and Panama serogroups. In addition, 12% showed lower MAT titers (50) for the same serogroups. Lastly, recently trapped feral squirrel monkeys were shown to have agglutinins against the Grippotyphosa and Sejroe serogroups. A vaccine, which was prepared from one of the strains isolated, was used in addition to antibiotic prophylaxis to control the enzootic disease. This confirms that the squirrel monkey is highly susceptible to icterohemorrhagic leptospirosis and is probably receptive to other serogroups, and that this animal may be useful in studying experimental leptospirosis and for testing new human vaccines.
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Spotted Fever Rickettsiae in Ticks from the Northern Sinai Governate, Egypt
Pages: 546–551More LessAbstractA field study was initiated in 1988 to investigate whether spotted fever group rickettsiae occur in geographic areas in Egypt that are adjacent to an area in the southern Israeli Negev that has a defined focus of spotted fever disease. Ticks were collected from dogs, sheep, and camels at four study sites in the northern Sinai. Tick hemolymph was processed for rickettsial detection by staining with fluorescein isothiocyanate-conjugated antibody to Rickettsia rickettsii. Of the 442 hemolymphs examined, 15 contained immunofluorescent rickettsiae. Eight hemolymph test-positive (HT +) ticks were Rhipicephalus sanguineus removed from dogs; the other HT + ticks comprised three Hyalomma species, H. anatolicum, H. impeltatum, and H. dromedarii. Both HT + and HT - ticks were tested for rickettsial DNA using the polymerase chain reaction (PCR). Eight of 10 HT + field-collected ticks were PCR positive (PCR +). All laboratory colony R. rickettsii-infected ticks were PCR +. No HT - ticks from field or laboratory isolates were PCR +.
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The Natural History of Endemic Trachoma: a Longitudinal Study
Pages: 552–559More LessAbstractA longitudinal study of trachoma was conducted among 100 members of nine families living in a hyperendemic area of Tanzania. Family members were examined for trachoma every three months for one year and conjunctival specimens were collected for antigen detection, which was performed either by direct fluorescent antibody cytologic analysis or enzyme immunoassay. The serovar specificity of tear antibodies was determined. Overall, young children tended to form a core of those with persistent, often severe, disease who consistently shed Chlamydia. These children are a potential source of infection in their family. Chlamydia could be identified early in the course of presumed recently acquired infections, but not later in resolving infections. This temporal change may account for the discrepancies between demonstrable organisms and clinical disease seen in cross-sectional studies. Several children were identified who did not develop trachoma despite having Chlamydia identified in conjunctival scrapings. Their ability to resist infection may offer clues for vaccine development. The study of serovar specificity is consistent with the intrafamily transmission of trachoma, but was confounded by the large family size and the potential for separate transmission units to occur within large extended families. These observations give further understanding of the natural history and kinetics of the transmission of trachoma that should be of use in developing and evaluating intervention studies.
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Epidemic Dermatitis due to Contact with a Moth in Cozumel, Mexico
Pages: 560–563More LessAbstractIn early December 1989, an outbreak that was initially thought to be scabies was investigated among employees of tourist hotels in Cozumel, Mexico. Of 417 employees interviewed, only 19 (4.6%) met a case definition for scabies dermatitis, while 91 (21.8%) reported a nonspecific dermatitis of less than one-week's duration. Persons with nonspecific dermatitis related the onset of their dermatitis to skin contact with a moth that had been present in large numbers in November. At the time of the initial investigation in December, there were no active cases of dermatitis and the moth was no longer present. During early January 1990, numerous cases of dermatitis again began to be reported. Using a case definition for nonspecific dermatitis, a survey of Cozumel's resident population showed an attack rate of 12.1%. A case-control study revealed the only significant risk factor to be skin contact with the suspect moth (P < 0.01), which had returned in large numbers. Six health workers volunteered to have the moth rubbed on their skin; within 5 min, five of six developed an intense pruritus followed by an erythematous rash. The moth was classified as Hylesia alinda Druce. This species has nettling hairs on its abdomen that excrete a histamine-like substance. Although this moth is normally present in small numbers in Cozumel, the passage of hurricane Gillbert kiled most of its natural predators (wasps and bees), allowing its population to overgrow. No control measures were undertaken because the moth's natural predators returned that spring and dramatically reduced the moth population. No further outbreaks of dermatitis occurred.
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High Prevalence of Hepatitis C Viremia among Aplastic Anemia Patients and Controls from Thailand
Pages: 564–570More LessAbstractAplastic anemia is a rare, life-threatening disease of unknown etiology, with unusually high prevalence in Thailand. It is sometimes associated with non-A, non-B hepatitis (NANBH). The hepatitis C virus (HCV), one of the causes of NANBH, is similar to flaviviridiae, a family of viruses many of whose members cause acute bone marrow suppression. To test the hypothesis that HCV viremia is associated with aplastic anemia among patients in Thailand, we compared 53 untransfused hospitalized aplastic anemia patients and 39 untransfused controls hospitalized for other conditions. We used the polymerase chain reaction to identify HCV viremia in three (5.7%) untransfused patients and two (5.1%) untransfused controls (P = 1.0, by Fisher's two-tailed exact test). Although our data do not exclude the possibility that a small subset of aplastic anemia cases are precipitated by HCV, we conclude that HCV viremia is not generally associated with aplastic anemia in Thailand. Our results also imply that the prevalence of HCV viremia may be unexpectedly high among untransfused persons in Thailand, a hypothesis that should be tested in other populations.
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Child Growth and Schistosomiasis Japonica in Northeastern Leyte, the Philippines: Cross-Sectional Results
Pages: 571–581More LessAbstractThe association between schistosomiasis japonica and child growth was studied cross-sectionally in 1, 561 males and females aged 4–19.9 years residing in an endemic region of northeastern Leyte, The Philippines. Stature, weight, upper arm muscle area, and sum of triceps and subscapular skinfold thicknesses were measured and related to presence of Schistosoma japonicum eggs in Kato stool smears and to the intensity of infection assessed by quantitative egg count. The presence of hookworm, ascaris, and trichuris eggs was also measured. Multivariable models were used to control for the effects of age, age2, and polyparasitism on growth. The prevalence of schistosomiasis was 31% in males and 22% in females, with the maximum prevalence in adolescence. In 8–19-year-old subjects, the intensity of schistosomiasis japonica was significantly related in males to reduced arm muscle area and sum of skinfolds (both P < 0.01) and in females to reduced stature, weight, arm muscle area, and sum of skinfolds (all P < 0.01). The greatest age-specific differences were during adolescence in both males and females. The growth retarding effects of intensity of schistosomiasis japonica were independent of the influence of other parasites, notably hookworm. Due to the magnitude of the schistosomiasis-associated growth differences in adolescence, adult body size, function and productivity may be affected.
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Plasma Pharmacokinetics and Therapeutic Efficacy of Praziquantel and 4-Hydroxypraziquantel in Schistosoma japonicum-Infected Rabbits after Oral, Rectal, and Intramuscular Administration
Pages: 582–588More LessAbstractThe relationship between plasma level and therapeutic efficacy of praziquantel (PZQ) and its major human oxidative metabolite, 4-hydroxypraziquantel (4-OHPZQ), has been investigated in Schistosoma japonicum-infected rabbits using three different routes of PZQ administration. After intramuscular administration (20 mg/kg), the maximum level of PZQ in rabbit cardiac plasma was 1.6 ± 1.0 µg/ml (mean ± SD) 30 min after administration. After oral or rectal administration (40 mg/kg), maximum plasma levels were 0.1 ± 0.2 µg/ml (oral) and 0.5 ± 0.4 µg/ml (rectal). The corresponding maximum 4-OHPZQ concentrations in cardiac plasma were 4.6 ± 1.8 µg/ml (intramuscular), 1.7 ± 0.5 µg/ml (oral), and 4.1 ± 1.6 µg/ml (rectal) 2 hr after administration of PZQ. After administration of similar doses, maximum levels of PZQ in plasma from the femoral vein were 29.3 ± 27.5 µg/ml (intramuscular), 0.6 ± 1.0 µg/ml (oral), and 0.7 ± 0.6 µg/ml (rectal). However, 60 min after intramuscular administration, the maximum PZQ concentration in portal venous blood was only 1.0 ± 0.6 µg/ml, which is substantially less than corresponding maximum portal vein levels after oral (6.8 ± 6.5 µg/ml) or rectal (3.7 ± 4.6 µg/ml) administration. Therapeutically, in spite of the 4–6-fold lower levels of PZQ in portal venous plasma after intramuscular administration, adult worm reduction rates in infected rabbits using the above doses were 92.2% (intramuscular), 90.1% (rectal), and 72.5% (oral), respectively, four weeks after treatment. Thus, no direct correlation between levels of PZQ in peripheral or portal venous blood and therapeutic efficacy was observed in rabbits infected with S. japonicum.
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Effect of Different Fractions of Heparin on Plasmodium falciparum Merozoite Invasion of Red Blood Cells in Vitro
Pages: 589–594More LessAbstractHeparin and various heparin fractions were separated according to differences in molecular weight or affinity for antithrombin III and used for the inhibition of Plasmodium falciparum merozoite invasion of red blood cells in vitro. No variation in sensitivity to heparin was found among the four strains of P. falciparum tested; all required approximately 5 µg/ml (0.5 U/ml) of heparin for 50% inhibition of invasion. The most efficient fraction of heparin was the one with low affinity for antithrombin III. Its 50% inhibition concentration was 1 µg/ml, indicating that it was more efficient than unfractionated heparin and other heparin fractions. The effect of heparin was reversible, since washing of heparin-treated cultures containing mainly schizonts showed no inhibition of merozoite invasion. The results suggest that a heparin fraction with no anticoagulant effect might be useful in the treatment of patients with falciparum malaria.
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Disruption of Plasmodium falciparum Erythrocyte Rosettes by Standard Heparin and Heparin Devoid of Anticoagulant Activity
Pages: 595–602More LessAbstractWe have studied the ability of heparin to disrupt spontaneous rosettes formed between Plasmodium falciparum-infected and uninfected red blood cells, which has been proposed to have importance in the pathogenesis of cerebral malaria. Substantial variation in this activity was found among six laboratory stains of P. falciparum. Rosettes formed by three of these strains were highly sensitive to heparin (50% disruption at 0.5–25 µg/ml; 1 µg/ml corresponds to 0.15 IU/ml). The rosettes formed by two other strains showed a much lower sensitivity (50% disruption at 700–2,500 µg/ml), while the rosettes formed by another strain were almost completely resistant to heparin (20% disruption at 6,500 µg/ml). The ability of heparin (65 or 650 µg/ml) to disrupt rosettes formed by 54 fresh Gambian isolates of P. falciparum also varied. Rosettes of 27 (50%) of the 54 isolates were disrupted to a significant degree (≥ 15%), while rosettes of the other 27 isolates remained unaffected at the concentrations tested. Heparin was fractionated by molecular weight and/or affinity for antithrombin III. We found that its property of rosette disruption was associated, to some extent, with size (high molecular weight) but not with its anticoagulant potential (affinity for antithrombin III). A heparin fraction with low affinity for antithrombin III and one with combined high molecular weight and low affinity for antithrombin III were as effective at disrupting rosettes as standard heparin, while a chemically modified (N-acetylated) high molecular weight-heparin fraction, similarly devoid of anticoagulant activity, lacked strong anti-rosette potential. The heparin fractions with low affinity for antithrombin III and high molecular weight/low affinity for antithrombin III may prove to be useful in the treatment of severe P. falciparum malaria.
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Use of the Falcon™ Assay Screening Test-Enzyme-Linked Immunosorbent Assay (FAST-ELISA) and the Enzyme-Linked Immunoelectrotransfer Blot (EITB) to Determine the Prevalence of Human Fascioliasis in the Bolivian Altiplano
Pages: 603–609More LessAbstractA collaborative study between the University of Puerto Rico School of Medicine, the Centers for Disease Control, the Bolivian Ministry of Health, and private voluntary organizations (Foster Parents Plan International and Danchurchaid) working in Bolivia has identified a region in the northwestern Altiplano of Bolivia near Lake Titicaca as harboring the highest prevalence of human fascioliasis in the world reported to date. Two serologic techniques (the Falcon™ assay screening test-enzyme-linked immunosorbent assay [FAST-ELISA] and the enzyme-linked immunoelectrotranfer blot [EITB]) were used in the determination of its prevalence. One hundred serum samples and 73 stool samples were obtained from Aymara Indians from Corapata, Bolivia. Antibody absorbance levels to Fasciola hepatica excretion-secretion antigens were compared with EITB banding patterns using the same antigen preparation. A positive FAST-ELISA result was defined as an absorbance value greater than the mean plus three standard deviations of two sets of normal negative controls (Puerto Rican and Bolivian). Using this criterion, 53 of 100 sera tested were found positive by this technique. Within this group, 19 (95%) of 20 individuals who were parasite positive were also positive by FAST-ELISA. An additional 24 individuals who were negative for F. hepatica eggs and 10 individuals for whom no specimens were received were also positive by FAST-ELISA. Among the 53 individuals negative for F. hepatica eggs, 29 were also negative by FAST-ELISA. The EITB analysis of the sera from confirmed infected individuals revealed at least three F. hepatica (Fh) bands with molecular weights of 12, 17, and 63 kD, respectively. All 20 sera from infected individuals recognized the Fh12 band; the Fh17 and Fh63 bands were only observed in those individuals with the highest FAST-ELISA absorbances, which suggests that they may be markers for acute infection, in which antibody levels tend to be high. Additional serum samples from six individuals that were negative by coprology and FAST-ELISA did not recognize any of the above markers. Using EITB, 42 (79%) of the 53 persons who were positive by FAST-ELISA were confirmed. These studies support the use of immunologic techniques in the determination of prevalence in epidemiologic studies of human fascioliasis.
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Immunodiagnosis of Human Cysticercosis (Taenia solium): a Field Comparison of an Antibody-Enzyme-Linked Immunosorbent Assay (ELISA), an Antigen-ELISA, and an Enzyme-Linked Immunoelectrotransfer Blot (EITB) Assay in Peru
Pages: 610–615More LessAbstractWe compared results of an enzyme-linked immunosorbent assay (ELISA) and an enzyme-linked immunoelectrotransfer blot (EITB) assay for the diagnosis of cysticercosis in sera and cerebrospinal fluid (CSF). Sera from 34 patients with confirmed cysticercosis were tested by both ELISA and EITB assays. Cerebrospinal fluid from some of these patients was also tested by ELISA for the presence of antibody (AB-ELISA) (n = 21) and antigen (AG-ELISA) (n = 15). Specificity in sera was examined by testing 51 serum samples from Bangladesh, where cysticercosis is not endemic. Cross-reactivity was evaluated in sera from patients with Echinococcus granulosus (hydatid) and Hymenolepis nana infections. Sensitivity in detecting cysticercosis in sera was 94% by EITB and 65% by AB-ELISA (P < 0.01). Sensitivities in the CSF tested by EITB, AB-ELISA, and AG-ELISA were 86%, 62%, and 67%, respectively. The specificity of the EITB was 100%, while that of AB-ELISA was 63% (P < 0.01). Cross-reactions occurred in the AB-ELISA with 11% and 20% of sera from hydatid and H. nana patients, respectively. Our results demonstrate that the EITB is the best assay available for the diagnosis of cysticercosis in both sera and CSF.
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An Improved Polymerase Chain Reaction Assay to Detect Trypanosoma cruzi in Blood
Pages: 616–623More LessAbstractAmplification by the polymerase chain reaction of Trypanosoma cruzi satellite DNA was used to enhance sensitivity in the detection of the parasite in blood, with the ultimate goal of improving diagnosis of the chronic phase of Chagas' disease. Two contiguous oligonucleotides were synthesized corresponding to the most conserved region of the 195-basepair repeated sequence and used as primers for the amplification reaction. Nineteen femtograms of parasite DNA that was amplified in the presence of 15 µg of human or mouse DNA produced a visible band upon electrophoresis in agarose gels and staining with ethidium bromide. In reconstitution experiments, one parasite in 10 ml of blood could be unambiguously determined when the DNA was isolated from nuclei after the blood was treated with NP40 and centrifuged. Polymerase chain reaction assays were carried out to detect T. cruzi in chronically infected mice. Most mice were parasite-positive when organs or tissues were tested, but all were negative when total blood was tested.
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