Standardization of dot-ELISA for the serological diagnosis of toxocariasis and comparision of the assay with ELISA

The dot-enzyme-linked immunosorbent assay (dot-ELISA) was standardized using somatic (S) and excretory-secretory (ES) antigens of Toxocara-canis for the detection of specific antibodies in 22 serum samples from children aged 1 to 15 years, with clinical signs of toxocariasis. Fourteen serum samples from apparently normal individuals and 28 sera from patients with other pathologies were used as controls. All samples were used before and after absorption with Ascaris suum extract. When the results were evaluated in comparison with ELISA, the two tests were found to have similar sensitivity, but dot-ELISA was found to be more specific in the presence of the two antigens studied. Dot-ELISA proved to be effective for the diagnosis of human toxocariasis, presenting advantages in terms of yield, stability, time and ease of execution and low cost.

Dot-ELISA for the detection of anti-Cysticercus cellulosae antibodies in cerebrospinal fluid using a new solid phase (resin-treated polyester fabric) and Cysticercus longicollis antigens

A dot-ELISA was developed for the detection of antibodies in CSF in the immunologic diagnosis of human neurocysticercosis, using antigen extracts of the membrane and scolex of Cysticercus cellulosae (M+S-Cc) and, alternately, membrane (M) and vesicular fluid (VF) of Cysticercus longicollis (Cl) covalently bound to a new solid phase consisting of polyester fabric treated with N-methylol-acrylamide resin (dot-RT). The test was performed at room temperature, with reduced incubation times and with no need for special care in the manipulation of the support. The sensitivity rates obtained were 95.1% for antigen Cc and 97.6% for antigen Cl. Specificity was 90.6% when Cc was used, and 96.9% and 100% when M-Cl and VF-Cl were used, respectively. No significant differences in titer were observed between tests carried out with homologous and heterologous antigens. The low cost and easy execution of the dot-RT test using antigen extracts of Cysticercus longicollis indicate the test for use in the immunodiagnosis of human neurocysticercosis.

Cryo-Microtome sections of coproculture larvae of Strongyloides stercoralis and Strongyloides ratti as antigen sources for the immunodiagnosis of human strongyloidiasis

Cryo-microtome sections of larvae of Strongyloides stercoralis and S. ratti respectively obtained from human and rat feces cultures, were used as antigens. Fluoresceinate conjugates against human IgG were employed at the ideal titer of 10 for S. stercoralis and 100 for S. ratti. The sensitivity of the indirect immunofluorescence reaction (IIF) was 94.4% and 92.5% and the specificity 94.2% and 97.1% for the two specific larval antigens, respectively. Sera from 123 persons (54 from carriers of S. stercoralis infections and 69 from controls) were submitted to the reaction. The titers of different sera varied from 20 to 2560. There was a significant linear correlation (r = 0.85 p £ 0.001) between the antibodies from the two species of larval antigens. We conclude that both antigens may be used in the IIF reaction for the diagnosis of human strongyloidiasis. Due to the feasibility of safe and low-cost mass production of S. ratti larvae in the laboratory with a considerable economy of conjugate, their utilization in the serum diagnosis of human strongyloidiasis is recommended

Microbicidal effect of medicinal plant extracts (Psidium guajava Linn. and Carica papaya Linn.) upon bacteria isolated from fish muscle and known to induce diarrhea in children

Out of the twenty-four samples of shrimp and fish muscle used for this study, twelve were collected near a large marine sewer for waste disposal, 3 km off the coast of Fortaleza (Brazil) and used for the isolation of E. coli. Other twelve were collected at the Mucuripe fresh fish market (Fortaleza, Brazil) and used for the isolation of Staphylococcus aureus. Ethanol, water and acetone-diluted extracts of guava and papaya leaf sprouts were tested on the bacteria in order to verify their microbicidal potential. The E. coli strains used in the trials were rated LT positive. The papaya leaf extracts (Carica papaya Linn) showed no microbicidal activity while the guava sprout extracts (Psidium guajava Linn) displayed halos exceeding 13 mm for both species, an effect considered to be inhibitory by the method employed. Guava sprout extracts by 50% diluted ethanol most effectively inhibited E. coli (EPEC), while those in 50% acetone were less effective. It may be concluded that guava sprout extracts constitute a feasible treatment option for diarrhea caused by E. coli or by S. aureus-produced toxins, due to their quick curative action, easy availability in tropical countries and low cost to the consumer.

Comparative study of the topical effectiveness of the andiroba oil (Carapa guianensis) and DEET 50% as repellent for Aedes sp

DEET (N,N-diethyl-3-methylbenzamide) is nowadays the most effective mosquito repellent available, however, its use can present some topical and systemic side effects. Some botanical compositions, as Andiroba (Carapa guianensis), have been proved repellent properties at low cost and toxicity. An experimental study was driven involving four volunteers submitting their forearms covered with Andiroba oil at 100%, DEET 50%, refined soy oil, Andiroba oil 15% and in the absence of products, directly to healthy females of Aedes sp. The times of first and third bites were checked. The results showed that the median of the first bite without any product was 17.5s and the third bite, 40.0s. In the soy oil, the bites happened in 60.0s and 101.5s, in the presence of Andiroba oil 100%, in 56.0s and 142.5s and in Andiroba oil 15%, in 63.0s and 97.5s. The volunteers using DEET 50% had not received bites after 3600s in most of the experiments (p < 0.001 Wilcoxon). Pure Andiroba oil compared to the soy oil, forearm without product and Andiroba oil 15%, showed discreet superiority (p < 0.001 Wilcoxon). Our conclusion is that this study demonstrated that the pure Andiroba oil presents discreet repellent effect against bite of Aedes sp., being significantly inferior to DEET 50%.

Reproduction of Leishmania (Leishmania ) infantum chagasi in conditioned cell culture growth medium

Leishmanias can be produced by inoculation in conditioned McCoy cell culture growth medium (CGM). Leishmania (Leishmania) infantum chagasi (100 parasites) grown in NNN medium was inoculated in 2.5 mL CGM, kept in plates (24 wells) and its multiplication was observed for five days (120 hours). After day 5, the medium was saturated with the flagellate forms of the parasite (promastigotes). The reproduction of the leishmanias was observed every 24 hours and the number of parasites was calculated by counting the parasites in a drop of 10 µ L and photomicrographied. So the number of Leishmanias was adjusted to 1 mL volume. The advantage of the technique by isolation of Leishmania in CGM demonstrated in this study is its low cost and high efficacy even with a small quantity of parasites (10² promastigotes) used as inoculum. Additionally, isolation of the leishmania can be obtained together with an increase in their density (180 times) as observed by growth kinetics, within a shorter time. These results justify the use of this low-cost technique for the isolation and investigation of the behavior and multiplication of Leishmania both in vertebrates and invertebrates, besides offering means of obtaining antigens, whether whole antigens (leishmanias) or the soluble antigens produced by the parasites which may be useful for the production of new diagnostic kits.

Impact of Taraxerol in combination with extract of Euphorbia tirucalli plant on biological parameters of Lymnaea acuminata

The present work was carried out to evaluate the molluscicidal activity of active ingredient Taraxerol with the acetone extract of Euphorbia tirucalli against Lymnaea acuminata snail. The (fecundity, hatchability and survivability) of snail L. acuminata exposed to this extract was studied. The effects of the tested extracts on life-history traits of harmful snail L. acuminata have also been evaluated, and this study also expounds the inhibitory effects of these extracts singly as well as in binary combination (1:1 ratio). It concluded that these herbal products act as a potential source of molluscicides, and that they would also have the advantage of easy availability, low cost, biodegradability and greater acceptance amongst users than synthetic pesticide.

THE SCHISTOSOMULA TEGUMENT ANTIGEN AS A POTENTIAL CANDIDATE FOR THE EARLY SEROLOGICAL DIAGNOSIS OF SCHISTOSOMIASIS MANSONI

If Schistosoma mansoni infection could be detected in its early stages, especially before the egg deposition in the host tissues, the development of severe pathologic lesions could be efficiently prevented. We therefore developed an indirect enzyme-linked immunosorbent assay based on the detection of specific IgG against schistosomula antigens (ELISA-SmTeg). The assay was applied in sera samples from non-infected and infected mice collected seven and 15 days post-infection. The results were compared to the number of adult worms obtained by perfusion of the murine hepatic system 50 days post-infection. The sensitivity and specificity of the ELISA-SmTeg were 100% (p = 0.0032 and 0.0048 respectively for seven and 15 days of infection) with a cutoff value of 0.15 (p = 0.0002). Our findings show a novel low-cost serological assay using antigens which are easy to obtain, which was able to detect all the infected mice as early as seven days post-infection.

Helminthiases in Montes Claros. Preliminary survey

A preliminary survey was conducted for the presence of helminths in the city of Montes Claros, M. G., Brazil. Three groups of persons were examined by the direct smear, Kato thick film and MIFC techniques; one group by direct smear and Kato only. General findings were: a high prevalence of hookworm, followed by ascariasis, S. mansoni, S. stercoralis and very light infections with T. trichiurá. E. vermicularis and H. nana were ranking parasites at an orphanage, with some hookworm and S. mansoni infections as well. At a pig slaughter house, the dominant parasites were hookworm and S. mansoni. Pig cysticercosis was an incidental finding worth mentioning for the health hazard it represents for humans as well as an economic loss. From the comparative results between the Kato and the MIF the former proved itself again as a more sensitive and reliable concentration method for helminth eggs, of low cost and easy performance.

Validation of a case definition for leptospirosis diagnosis in patients with acute severe febrile disease admitted in reference hospitals at the State of Pernambuco, Brazil

INTRODUCTION: Leptospirosis is often mistaken for other acute febrile illnesses because of its nonspecific presentation. Bacteriologic, serologic, and molecular methods have several limitations for early diagnosis: technical complexity, low availability, low sensitivity in early disease, or high cost. This study aimed to validate a case definition, based on simple clinical and laboratory tests, that is intended for bedside diagnosis of leptospirosis among hospitalized patients. METHODS: Adult patients, admitted to two reference hospitals in Recife, Brazil, with a febrile illness of less than 21 days and with a clinical suspicion of leptospirosis, were included to test a case definition comprising ten clinical and laboratory criteria. Leptospirosis was confirmed or excluded by a composite reference standard (microscopic agglutination test, ELISA, and blood culture). Test properties were determined for each cutoff number of the criteria from the case definition. RESULTS: Ninety seven patients were included; 75 had confirmed leptospirosis and 22 did not. Mean number of criteria from the case definition that were fulfilled was 7.8±1.2 for confirmed leptospirosis and 5.9±1.5 for non-leptospirosis patients (p<0.0001). Best sensitivity (85.3%) and specificity (68.2%) combination was found with a cutoff of 7 or more criteria, reaching positive and negative predictive values of 90.1% and 57.7%, respectively; accuracy was 81.4%. CONCLUSIONS: The case definition, for a cutoff of at least 7 criteria, reached average sensitivity and specificity, but with a high positive predictive value. Its simplicity and low cost make it useful for rapid bedside leptospirosis diagnosis in Brazilian hospitalized patients with acute severe febrile disease.

A comparison of four DNA extraction protocols for the analysis of urine from patients with visceral leishmaniasis

Introduction Polymerase chain reaction (PCR) may offer an alternative diagnostic option when clinical signs and symptoms suggest visceral leishmaniasis (VL) but microscopic scanning and serological tests provide negative results. PCR using urine is sensitive enough to diagnose human visceral leishmaniasis (VL). However, DNA quality is a crucial factor for successful amplification. Methods A comparative performance evaluation of DNA extraction methods from the urine of patients with VL using two commercially available extraction kits and two phenol-chloroform protocols was conducted to determine which method produces the highest quality DNA suitable for PCR amplification, as well as the most sensitive, fast and inexpensive method. All commercially available kits were able to shorten the duration of DNA extraction. Results With regard to detection limits, both phenol: chloroform extraction and the QIAamp DNA Mini Kit provided good results (0.1 pg of DNA) for the extraction of DNA from a parasite smaller than Leishmania (Leishmania) infantum (< 100fg of DNA). However, among 11 urine samples from subjects with VL, better performance was achieved with the phenol:chloroform method (8/11) relative to the QIAamp DNA Mini Kit (4/11), with a greater number of positive samples detected at a lower cost using PCR. Conclusion Our results demonstrate that phenol:chloroform with an ethanol precipitation prior to extraction is the most efficient method in terms of yield and cost, using urine as a non-invasive source of DNA and providing an alternative diagnostic method at a low cost.

Evaluation of the reference value for the Montenegro skin test

Abstract:INTRODUCTION:The Montenegro skin test (MST) has good clinical applicability and low cost for the diagnosis of American tegumentary leishmaniasis (ATL). However, no studies have validated the reference value (5mm) typically used to discriminate positive and negative results. We investigated MST results and evaluated its performance using different cut-off points.METHODS:The results of laboratory tests for 4,256 patients with suspected ATL were analyzed, and 1,182 individuals were found to fulfill the established criteria. Two groups were formed. The positive cutaneous leishmaniasis (PCL) group included patients with skin lesions and positive direct search for parasites (DS) results. The negative cutaneous leishmaniasis (NCL) group included patients with skin lesions with evolution up to 2 months, negative DS results, and negative indirect immunofluorescence assay results who were residents of urban areas that were reported to be probable sites of infection at domiciles and peridomiciles.RESULTS:The PCL and NCL groups included 769 and 413 individuals, respectively. The mean ± standard deviation MST in the PCL group was 12.62 ± 5.91mm [95% confidence interval (CI): 12.20-13.04], and that in the NCL group was 1.43 ± 2.17mm (95% CI: 1.23-1.63). Receiver-operating characteristic curve analysis indicated 97.4% sensitivity and 93.9% specificity for a cut-off of 5mm and 95.8% sensitivity and 97.1% specificity for a cut-off of 6mm.CONCLUSIONS:Either 5mm or 6mm could be used as the cut-off value for diagnosing ATL, as both values had high sensitivity and specificity.

Diagnosis of latent Mycobacterium tuberculosis infection: tuberculin test versus interferon-gamma release

Abstract: INTRODUCTION: The treatment of individuals with active tuberculosis (TB) and the identification and treatment of latent tuberculosis infection (LTBI) contacts are the two most important strategies for the control of TB. The objective of this study was compare the performance of tuberculin skin testing (TST) with QuantiFERON-TB Gold In TUBE(r) in the diagnosis of LTBI in contacts of patients with active TB. METHODS: Cross-sectional analytical study with 60 contacts of patients with active pulmonary TB. A blood sample of each contact was taken for interferon-gamma release assay (IGRA) and subsequently performed the TST. A receiver operating characteristic curve was generated to assess the cutoff points and the sensitivity, predictive values, and accuracy were calculated. The agreement between IGRA and TST results was evaluated by Kappa coefficient. RESULTS: Here, 67.9% sensitivity, 84.4% specificity, 79.1% PPV, 75% NPV, and 76.7% accuracy were observed for the 5mm cutoff point. The prevalence of LTBI determined by TST and IGRA was 40% and 46.7%, respectively. CONCLUSIONS: Both QuantiFERON-TB Gold In TUBE(r) and TST showed good performance in LTBI diagnosis. The creation of specific diagnostic methods is necessary for the diagnosis of LTBI with higher sensitivity and specificity, preferably with low cost and not require a return visit for reading because with early treatment of latent forms can prevent active TB.

Omeprazole, Furazolidone, and Tetracycline: an eradication treatment for resistant H. pylori in Brazilian patients with peptic ulcer disease

OBJECTIVES: To determine the efficacy of a simple, short-term and low-cost eradication treatment for Helicobacter pylori (H. pylori) using omeprazole, tetracycline, and furazolidone in a Brazilian peptic ulcer population, divided into 2 subgroups: untreated and previously treated for the infection. PATIENTS AND METHODS: Patients with peptic ulcer disease diagnosed by endoscopic examination and infected by H. pylori diagnosed by the rapid urease test (RUT) and histological examination, untreated and previously unsuccessfully treated by macrolides and nitroimidazole, were medicated with omeprazole 20 mg daily dose and tetracycline 500 mg and furazolidone 200 mg given 3 times a day for 7 days. Another endoscopy or a breath test was performed 12 weeks after the end of treatment. Patients were considered cured of the infection if a RUT and histologic examination proved negative or a breath test was negative for the bacterium. RESULTS: Sixty-four patients were included in the study. The women were the predominant sex (58%); the mean age was 46 years. Thirty-three percent of the patients were tobacco users, and duodenal ulcer was identified in 80% of patients. For the 59 patients that underwent follow-up examinations, eradication was verified in 44 (75%). The eradication rate for the intention-to-treat group was 69%. The incidence of severe adverse effects was 15%. CONCLUSION: The treatment provides good efficacy for H. pylori eradication in patients who were previously treated without success, but it causes severe adverse effects that prevented adequate use of the medications in 15% of the patients.

Improving Post-Discharge Medication Adherence in Patients with CVD: A Pilot Randomized Trial

Background:Effective interventions to improve medication adherence are usually complex and expensive.Objective:To assess the impact of a low-cost intervention designed to improve medication adherence and clinical outcomes in post-discharge patients with CVD.Method:A pilot RCT was conducted at a teaching hospital. Intervention was based on the four-item Morisky Medication Adherence Scale (MMAS-4). The primary outcome measure was medication adherence assessed using the eight-item MMAS at baseline, at 1 month post hospital discharge and re-assessed 1 year after hospital discharge. Other outcomes included readmission and mortality rates.Results:61 patients were randomized to intervention (n = 30) and control (n = 31) groups. The mean age of the patients was 61 years (SD 12.73), 52.5% were males, and 57.4% were married or living with a partner. Mean number of prescribed medications per patient was 4.5 (SD 3.3). Medication adherence was correlated to intervention (p = 0.04) and after 1 month, 48.4% of patients in the control group and 83.3% in the intervention group were considered adherent. However, this difference decreased after 1 year, when adherence was 34.8% and 60.9%, respectively. Readmission and mortality rates were related to low adherence in both groups.Conclusion:The intervention based on a validated patient self-report instrument for assessing adherence is a potentially effective method to improve adherent behavior and can be successfully used as a tool to guide adherence counseling in the clinical visit. However, a larger study is required to assess the real impact of intervention on these outcomes.