Immunoenzyme assay using micro Dot on nitrocellulose (Dot-ELISA) in the diagnosis of Chagas' disease. I. Comparative study of 2 antigenic preparations of Trypanosoma cruzi

Using the Dot-ELISA technique, two antigenic preparations of Trypanosoma cruzi epimastigote forms have been compared for the diagnosis of Chagas' disease: (1) The cytoplasmic fraction (cytoplasmic antigen) and (2) whole formalin fixed epimastigotes (integral antigen). There was been used sera from 95 chagasic patients with chronic cardiomyopathy, positive conventional serology and either positive or negative xenodiagnosis; 74 subjects with negative conventional serology, and either clinically normal or presenting cardiomyopathy; 74 patients with different diseases including syphilis, toxoplasmosis, leishmaniasis or autoantibodies such as rheumatoid factor and antinuclear antibodies. By defining the diagnostic titers (cut off): 1:512 for cytoplasmic antigen and 1:128 for the integral antigen, a sensitivity of 100% has been obtained with both antigenic preparations, being the specificity of 96% for the former and 100% for the latter when leishmaniasis sera were not included. A comparative study with conventional serology was carried out using 147 sera from a Laboratory of Chagas' diagnosis; Dot-ELISA with cytoplasmic antigen showed co-positivity index of 1.0, co-negativity 0.989 and efficiency of 0.993, and Dot-ELISA with integral antigen 1.0, 0.979 and 0.986 respectively. According to this evaluation, Dot-ELISA using whole formalin fixed epimastigotes might be a practical alternative for the serological diagnosis of Chagas' disease.     

High infection rates of Triatoma dimidiata are associated with low levels of Trypanosoma cruzi seroprevalence in Pedro Carbo, Ecuador. Use of a tc24 gene-based PCR approach

In control programs for vectorial transmission of Chagas' disease, conventional microscopic procedures are generally performed to determine baseline levels of infectivity of vectors. Reported here are data using Polymerase Chain Reaction in the detection of Trypanosoma cruzi in Triatoma dimidiata, one of the principal vectors of Chagas' disease in Ecuador. The microscopy and PCR techniques showed a high percentage of vector infection in Pedro Carbo, province of Guayas (Ecuador), with 44.16% and 46.13% positive insects, respectively. This contrasted with the very low Chagas seropositivity recorded (0.5%). Since T. dimidiata was the only vector of the Chagas' disease found in Pedro Carbo and looking at the vector behavior, our data suggest that despite the high T. dimidiata infection, the low Chagas seropositivity detected is closely associated with the epidemiological and ecological context of T. dimidiata in Pedro Carbo.     

Leptospiral glycolipoprotein as a candidate antigen for serodiagnosis of human leptospirosis

Aims:  Development of a simple, specific, rapid and inexpensive Dot-ELISA test for early diagnosis of human leptospirosis.
Methods and Results:  Serum samples from 90 patients diagnosed with leptospirosis were analysed by Dot-ELISA test incorporating Glycolipoprotein (GLP) antigen from serovars Copenhageni and Patoc. Results were compared with those obtained with microscopic agglutination test, currently, the gold standard reference serological method. Serum samples from healthy blood bank donors and patients diagnosed with diseases other than leptospirosis were used as negative controls. The specificities of both GLP-based assays were 97·1% and 100% with serum samples from patients with other diseases and with serum samples from healthy control group, respectively. With serum samples from patients with acute leptospirosis, sensitivity was 76·6% with Dot-ELISA Copenhageni and 90·0% with Dot-ELISA Patoc. With serum samples from patients in convalescence, sensitivity was 100% with both GLP-based assays.
Conclusions:  This Dot-ELISA provides a candidate antigen for serodiagnosis of leptospirosis during all phases of illness and could be a good alternative method for the early diagnosis of leptospirosis.
Significance and Impact of the Study:  The Dot-ELISA test is simple, specific, rapid and inexpensive. It is suitable for identifying a large number of samples and, hence, reducing the death rate of patients with leptospirosis.     

Follow-up of patent and subpatent parasitemias and development of muscular lesions in mice inoculated with very small numbers of Trypanosoma cruzi.

A sequential analysis of patent and subpatent parasitemias, mortality, and histopathology during acute Chagas' disease experimentally produced by inoculation of 10 or 100 bloodstream forms of Trypanosoma cruzi Y strain in susceptible mice was carried out. Parasites were searched for comparatively using three different methods: direct counting, Ficoll-MI density flotation, and hemoculture. Ficoll-MI density flotation promptly discriminated with high reproducibility subpatent parasitemic states not detected in the blood samples analyzed by direct counting. Despite the high proportion of supposedly uninfected animals and depending on the postinfection time, the majority of the mice had bloodstream parasites at the subpatent level detected by Ficoll-MI, and all of them had muscular lesions during the acute phase. All Ficoll-MI-negative blood samples from infected mice were also negative by hemoculture. Normal mouse blood purposely contaminated with parasite quantities ranging from 200 to 2000/ml was tested comparatively by density flotation and hemoculture and showed frequencies of reisolation varying from 25 to 100%. Overall, these data showed that inoculum as low as 10 infective forms of Y strain is able to induce acute Chagas' disease in susceptible mice and that a subpatent parasitemic state of 600-1000 forms/ml is a common finding. The use of Ficoll-MI to detect subpatent parasitemia is discussed.     

Dot-ELISA检测莱姆病血清抗体的实验研究

经实验研究,建立了检测莱姆病血清IgG的Dot-ELISA方法。阻断试验表明,本法具有特异性。经对10例伯氏疏螺旋体人工感染兔血清和24例正常兔血清标本的检测证实,该法与常规ELISA法检测的符合率为100％。重复性试验表明其重复性良好。     

Transplacental antibodies. Part III: Maternal antibodies against polioviruses, M. parotitidis and M. parainfluenzae

In 1981 we examined 247 sera for the presence of antibodies against all three types of poliovirus and 253 sera for antibodies against M. parotitidis and three types of M. parainfluenzae viruses. The sera were obtained from the cord blood of mothers between 15 and 34 years of age. All mothers were divided into four age groups, each with primipara and multipara subgroups. The rate of seropositivity for type 1 and type 2 poliovirus-specific antibody was in all age groups higher than 90%, the overall seropositivity rate for type 3 poliovirus antibody was 83.6%, with 73.1% as the lowest rate for age group of youngest mothers. Significant seropositivity variations between the primipara and multipara subgroups were recorded only for type 3 antibody in the two age groups of oldest mothers (25-29 and 30-34 years). This is consistent with the assumed booster effect of Sabin vaccine strains on mothers of families with more than one child. Antibodies specific to mumps virus were present in the cord blood of 78.6% of all mothers and the rates of seropositivity were found to rise with the increasing age. Seropositivity for M. parainfluenzae type 1-specific antibody was demonstrated in 95.8%, for type 2-specific antibody in 98.9% and for type 3-specific antibody in 100% of mothers, which is suggestive of high herd immunity levels in the population.     

Morbidity in Chagas' disease. IV. Longitudinal study of 10 years in Pains and Iguatama, Minas Gerais, Brazil

An evolutive study of the "case-control" type was carried out in an endemic area of Chagas' disease in Minas Gerais State, Brazil, using two cross-section evaluations with an interval of ten years between them (1974-1984). Patients were paired for sex and age. In the first cross-section study 264 pairs one with a positive serology and the other with a negative serology for T. cruzi antibodies were included. In the second evaluation, ten years later, 235 patients among those with previous positive serology and 216 with negative serology were located, but only 110 pairs could be recomposed and reexamined (clinical examination, ECG and Rx of the heart and esophagus). The incidence of chagasic cardiopathy in the cases with positive serology but previously assymptomatic was 38.3% during the ten year period. On the other hand there was a deterioration in 24% of the patients with chagasic cardiopathy since the first examination. Considering all clinical forms of the disease in 34.5% of the patients the clinical situation deteriorated, in 57.3% there was no change and in 8.2% the situation improved. The general mortality in the period was 23% in the chagasic group and 10.6% in the control group, but the lethality by cardiopathy was 17% in chagasic group and only 23.3% in the control group. The mortality was twice as high in males than in females, mainly in the age group from 30 to 59 years.     

Responses of Anopheles gambiae complex mosquitoes to the use of untreated bednets in The Gambia

Population dynamics of the Anopheles gambiae complex of malaria vector mosquitoes were studied in four small hamlets in The Gambia. Bednets were used to reduce man/vector contact in two of the hamlets. High densities of An. gambiae, sensu lato, were present for only 3-8 weeks during the rainy season, depending on the position of the hamlet within the study area. The proportions of blood-fed mosquitoes caught indoors (83.0%) and existing from houses (11.6%) were lower in hamlets where bednets were used than in hamlets without (96.5% and 33.1% respectively). Fewer of the blood-fed mosquitoes had fed on man in houses where people slept under bednets (68.2%) than in those without (81.5%). However, the average number of infective bites received by children was still greater than one a year in hamlets where bednets were used. Consequently bednets are considered unlikely to be an effective malaria control measure so long as they are untreated with insecticide.     

Immunological and structural characterization of an epitope from the Trypanosoma cruzi KMP-11 protein

The K1 peptide is an HLA-A*0201-restricted cytotoxic epitope derived from the Trypanosoma cruzi KMP-11 protein, this being the etiological agent of Chagas' disease. This work describes the K1 peptide's secondary structure and its recognition by sera from chagasic patients. Circular dichroism and NMR spectroscopy analysis revealed that the K1 peptide adopts an alpha-helical conformation. Fifty-six percent of individuals had anti-K1 and 86% anti-KMP-11 antibodies by ELISA in the chronic Chagas' group and 28 and 68% in the indeterminate Chagas' group, respectively. By contrast, no reactivity was observed in sera from healthy individuals and tuberculosis patients. Antibody response subclass specificity to the K1 peptide was IgG1 and IgG3. Taken together these results support the idea that the K1 peptide acts as a B-cell-inducer epitope during Chagas' disease.     

HLA and beta-myosin heavy chain do not influence susceptibility to Chagas disease cardiomyopathy

An inflammatory dilated cardiomyopathy occurs in 30% of Chagas' disease patients, chronically infected by Trypanosoma cruzi, while the remaining infected individuals are asymptomatic. Studies have indicated a role for genetic factors in the susceptibility to Chagas' disease cardiomyopathy. In an attempt to identify the genetic factors influencing the development and outcome of Chagas' cardiomyopathy, we compared the frequencies of alleles from two candidate gene loci, class II HLA and a microsatellite marker for the human cardiac beta-myosin heavy chain gene in different clinical groups. Patients were grouped as asymptomatic or with severe or mild cardiomyopathy. The results indicate that the HLA and myosin microsatellite allele profiles in all cardiomyopathy and in asymptomatic groups are similar. In conclusion, these results establish that polymorphism of HLA-DR and -DQ molecules, as well as beta-cardiac myosin, do not influence the susceptibility to different clinical forms of Chagas' disease or the progression to severe Chagas' cardiomyopathy. On the other hand, male sex was identified as a risk factor for progression to the more severe forms of cardiomyopathy (relative risk = 8.75).     

Trypanosoma cruzi reinfections in mice determine the severity of cardiac damage

In two murine models we studied Trypanosoma cruzi reinfection in the acute and chronic phase of experimental Chagas' disease in order to elucidate the relevance of reinfections in determining the variability of cardiac symptoms and the irreversible cardiac damage. They were followed for 120 and 600 days post infection (p.i.) for the acute and chronic model, respectively. Reinfected mice reached higher parasitaemia levels than infected mice. The survival was 33 and 21% in the chronic phase for mice reinfected in the acute phase and 13% for mice reinfected in the chronic stage at the end of the experiments. Sixty-six percent of the infected group presented electrocardiographic abnormalities (heart frequency, prolonged PQ segment or QRS complex) in the chronic stage whereas 100% of the reinfected animals exhibited electric cardiac dysfunction since 90 and 390 days p.i. for the acute and chronic reinfected model, respectively (P<0.01). Heart histopathological studies showed fibrosis and necrosis areas and mononuclear infiltrates supporting the view that parasite persistence is a major factor in continuing the tissue inflammation. This work shows that T. cruzi reinfections could be related to the variability and severity of the clinical course of Chagas' disease and that parasite persistence is involved in exacerbation of the disease.     

Impact of permethrin-treated bednets on malaria transmission by the Anopheles gambiae complex in The Gambia

Malaria vector mosquitoes belonging to the Anopheles gambiae complex were studied in four hamlets in The Gambia. All inhabitants were given bednets treated either with a placebo (milk) in two hamlets or with the pyrethroid insecticide permethrin (500 mg/m2) in two other hamlets. Malaria transmission occurred mainly during a few weeks of the rainy season, in September and October 1987. The indoor resting densities of mosquitoes in permethrin-treated hamlets were reduced, and we estimated over 90% reduction in biting on man by An. gambiae Giles sensu stricto in these hamlets. No mosquitoes were found under permethrin-treated bednets compared with eighty-one recovered from placebo-treated bednets. Mosquitoes exited more readily from rooms where permethrin-treated bednets were used than from rooms with placebo-treated nets. The annual mean probability that a child would receive an infective bite was estimated to be 0.09 in hamlets with insecticide-treated bednets, compared with 1.9 where placebo-treated bednets were used. Permethrin-treated bednets are therefore recommended as a means of effectively reducing the risk of exposure to malaria transmission, particularly in areas of low seasonal transmission.     

Epidemiological status of Chagas disease in the endemic area from Region II of Antofagasta]

During 1997 a seroepidemiological study on Chagas' disease was carried out in 18 localities of three provinces (Tocopilla, El Loa and Antofagasta) of Region II (20 degrees 56'-26 degrees South Lat.; 70 degrees 38'-67 degrees West Long.), in order to assess the impact of the control program against Triatoma infestans launched in 1988, based on insecticide spraying of dwellings. By means of ELISA and an indirect hemagglutination test for Chagas' disease blood samples from 1,034 children under 10 years of age were examined, arising a 0.5% (3 cases) positivity. Test resulted positive in 2 (0.9%) children from the locality of San Pedro de Atacama and 1 (0.4%) from Calama city, all in the age group 6-10 year-old. However, none of their dwellings were found infested with T. infestants. These results indicate that the control program has a good possibility to prevent new human infections. It is advisable to continue the seroepidemiological and entomological vigilance and remark the necessity of increasing the effort in the study of transmission through other routes, to adopt or reinforce the pertinent preventive measures.     

Evaluation of the rabbit as a model for Chagas' disease. I. Parasitological studies

In order to investigate the value of the rabbit as an experimental model for Chagas' disease, 72 animals have been inoculated by intraperitoneal and conjunctival route with bloodstream forms, vector-derived metacyclic trypomastigotes and tissue culture trypomastigotes of Trypanosoma cruzi strains Y, CL and Ernane. In 95.6% of the animals trypomastigotes had been detected at the early stages of infection by fresh blood examination. The course of parasitemia at the acute phase was strongly influenced by the parasite strain and route of inoculation. At the chronic phase parasites had been recovered by xenodiagnosis and/or hemoculture in 40% of the examined animals. The xenodiagnosis studies have shown selective interactions between the T. cruzi strains and the four species of vectors used, inducing significant variability in the results. The data herein present are consistent with the parasitological requirements established for a suitable model for chronic Chagas' disease.     

Isolation of Trypanosoma cruzi samples by xenodiagnosis and hemoculture from patients with chronic Chagas' disease

Fifty nine chronic chagasic patients were simultaneously submitted to xenodiagnosis and hemoculture for Trypanosoma cruzi samples isolations. The xenodiagnosis was done with 40 Panstrongylus megistus, Triatoma infestans and Dipetalogaster maximus nymphs, performing 120 triatomines. Groups of 10 insects per specie were dissecated and the intestinal content pooled and examined, after previous trituration and homogenization. The microscopically negative material was seed into LIT medium and examined after 20 days. Twenty nine patients were parasitologically proved, being 15 only by xenodiagnosis, 4 only by hemoculture and 10 by both methods. It was discussed the parasitological comprovation difficulties in chronic chagasic patients, the value of the simultaneous utilization of different triatomine species in xenodiagnosis and the hemoculture, in a favorable positive association to the sensitivity increase in the diagnosis' disease. The 49.2% of positivity obtained in this group, visualize approaches like clinic-therapeutic assay and or epidemiological (case-control) with the purpose to investigate a possible association with T. cruzi samples and different clinic forms in Chagas' disease.     

After the epidemic: follow up study of HIV seroprevalence and changing patterns of drug use.

OBJECTIVE--To follow up known intravenous drug users to determine current health state and drug use, compare characteristics with those of recent drug users, and examine HIV exposure and serostate. DESIGN--Subjects were identified from conventional general practice records and recruited from 1980 to the end of 1985; they were followed up during 1987 and 1988 and compared with drug users identified in the same way but recruited after 1985. SETTING--General practice and community in north west Edinburgh. Follow up conducted throughout the United Kingdom. SUBJECTS--Subjects known to have injected illegal drugs before 1986 (n = 203) and since that time. MAIN OUTCOME MEASURES--Mortality from and prevalence of HIV seropositivity and various parameters indicative of abstinence. RESULTS--Of the 203 subjects in the follow up group, 189 (93%) were traced; 16 (8%) had died and the remaining 173 (85%) were interviewed. In all, 146 (72% of the follow up cohort) had been tested for HIV antibodies, 94 (64%) having positive and 52 (36%) negative results; 57 (28%) had not been tested. Of the 65 subjects in the recently recruited group, 51 (79%) had been tested for HIV, 15 (29%) having positive results. A further 21 (43%) were currently negative for HIV antibody but still at risk. Thirty three (19%) of those followed up were confirmed abstinent, although more (about half) showed evidence of diminished drug injecting. Age correlated strongly with abstinence (p less than 0.001). One third of the group currently used cannabis, buprenorphine, dihydrocodeine, or diazepam. When the two groups were analysed together there was a strong association between the date of starting injecting and HIV seropositivity (chi 3 = 23.81, df = 2, p less than 0.001), with a peak around 1980-3. CONCLUSIONS--Although only a fifth of the followed up group were convincingly abstinent, a much larger group showed evidence of prolonged periods of remission. Overall, much use of oral drugs was confirmed and worrying trends towards taking buprenorphine and benzodiazepines were evident. The peak incidence of starting drug use and the comparatively low seroprevalence of HIV in the newer drug users probably explain the anomalous high seroprevalence in Edinburgh drug users during 1980-5. The epidemic of HIV during the first half of the 1980s in the group suggests that the virus was probably being transmitted because of a pattern of behaviour. Changing patterns of HIV transmission suggest a need to concentrate on heterosexual transmission as the main problem in the future.     

No Proof of HTLV-I/II in Intravenous Drug Abusers with a High Rate of HIV-1 Infection in Central Thailand

Serum specimens of 1,074 intravenous drug abusers (IVDA) were examined for infection with HIV-1, HTLV-I and HTLV-II in central Thailand. Three hundred and sixty-two of the specimens were seropositive for HIV-1 (33.7%). The HIV-1 seropositive IVDAs exhibited increased seropositivity with age through group 40–44 and significantly decreased seropositivity over the age of 45. In contrast, no seropositivity to either HTLV-I or -II was detected in the samples tested by a particle-agglutination assay for HTLV followed by type-specific Western blotting for HTLV. Reference to previous reports suggested that the rate of HIV infection in IVDAs has decreased with no HTLV-I or HTLV-II in Thailand when compared with that of the HIV infection in 1992.     

Haemagglutination-inhibiting antibodies to B.K. virus in Hungary.

Antibody inhibiting the haemagglutination by B.K. virus was found in 64% of the serum samples collected from 949 subjects in Hungary. The frequency of seropositivity was the lowest (17%) among infants 1 and 2 years of age and the highest (93%) in the age group 16 to 20 years. The subsequent age groups showed a slow continuous decline. The age-distribution curve drawn on the basis of the geometric means of titres has two peaks, one between 3 and 5 years of age and another between 21 and 30 years of age, reaching 6.74 and 7.05, respectively, as expressed in terms of log2 units. The prevalence of haemagglutination-inhibiting antibody to B.K. virus in Hungary and its distribution by age are similar to those reported from other European countries.     

High prevalence anti-Trypanosoma cruzi antibodies,among blood donors in the State of Puebla,a non-endemic area of Mexico

Blood transfusion is the second most common transmission route of Chagas disease in many Latin American countries. In Mexico, the prevalence of Chagas disease and impact of transfusion of Trypanosoma cruzi-contaminated blood is not clear. We determined the seropositivity to T. cruzi in a representative random sample, of 2,140 blood donors (1,423 men and 647 women, aged 19-65 years), from a non-endemic state of almost 5 millions of inhabitants by the indirect hemagglutination (IHA) and enzyme linked immunosorbent assay (ELISA) tests using one autochthonous antigen from T. cruzi parasites, which were genetically characterized like TBAR/ME/1997/RyC-V1 (T. cruzi I) isolated from a Triatoma barberi specimen collected in the same locality. The seropositivity was up to 8.5% and 9% with IHA and ELISA tests, respectively, and up to 7.7% using both tests in common. We found high seroprevalence in a non-endemic area of Mexico, comparable to endemic countries where the disease occurs, e.g. Brazil (0.7%), Bolivia (13.7%) and Argentina (3.5%). The highest values observed in samples from urban areas, associated to continuous rural emigration and the absence of control in blood donors, suggest unsuspected high risk of transmission of T. cruzi, higher than those reported for infections by blood e.g. hepatitis (0.1%) and AIDS (0.1%) in the same region.     

Dot-ELISA法检测血液及唾液HIV抗体的应用评价

评价人类免疫缺陷病毒1+2型抗体检测试剂盒(Dot-ELISA法)检测血清和唾液样本的临床性能。采用对照试验研究,选取背景清晰的研究对象200例,采集同一研究对象的血清和唾液样本,应用万泰生物药业公司生产的人类免疫缺陷病毒1+2型抗体检测试剂盒作为考核试剂,法国生物梅里埃公司生产的人类免疫缺陷病毒抗体诊断试剂盒(ELISA法)作为参考试剂,考核试剂检测结果与参考试剂及研究对象背景进行比较分析。考核试剂检测血清HIV抗体与参考试剂相比较,阳性符合率100%,阴性符合率100%,总符合率100%,Kappa值1.00,一致性为最强;考核试剂检测唾液HIV抗体与参考试剂检测结果相比较,阳性符合率98.78%,阴性符合率100%,总符合率99.50%,Kappa值0.99,一致性为最强。Dot-ELISA法人类免疫缺陷病毒1+2型抗体检测试剂盒对血清及唾液样本检测性能优越,适合HIV抗体快速筛查。