Evaluation of a direct immunofluorescent antibody (difma) test using Leishmania genus - specific monoclonal antibody in the routine diagnosis of cutaneous leishmaniasis

A direct immunofluorescent antibody (DIFMA) test using a Leishmania genus- specific monoclonal antibody was evaluated in the routine diagnosis of cutaneous leishmaniasis (CL) in Ecuador. This test was compared with the standard diagnostic techniques of scrapings, culture and histology. Diagnostic samples were taken from a total of 90 active dermal ulcers from patients from areas of Ecuador known to be endemic for cutaneous leishmaniasis. DIFMA was positive in all lesions. It was shown to be significantly superior to standard diagnostic methods either alone or in combination. The sensitivity of DIFMA did not diminish with chronicity of lesions. This test proved to be extremely useful in the routine diagnosis of CL because it is highly sensitive, is easy to use and produces rapid results.

Evaluation of a commercial test based on ligase chain reaction for direct detection of Mycobacterium tuberculosis in respiratory specimens

A ligase chain reaction DNA amplification method for direct detection of Mycobacterium tuberculosis (Abbott LCx MTB) in respiratory specimens was evaluated. Results from LCx MTB Assay were compared with those from acid fast bacilli smear, culture, and final clinical diagnosis for each patient. A total of 297 respiratory specimens (sputum and bronchial lavage) from 193 patients were tested. The sensitivity, specificity, positive predictive value and negative predictive value of LCx vs culture were 92.7%, 93%, 67.8% and 98.7%, respectively. When compared to the clinical final diagnosis, the sensitivity, specificity, PPV and NPV for LCx were 88.9%, 96.8%, 86.5% and 97.4%, respectively. The sensitivity of LCx MTB assay was 75% for smear-negative, culture positive samples. The results indicate that LCx MTB assay is a rapid, simple and valuable technique as a complementary tool for the diagnosis of tuberculosis.

4-Second Exercise Test: Reference Values for Ages 18–81 Years

Background: Physiological reflexes modulated primarily by the vagus nerve allow the heart to decelerate and accelerate rapidly after a deep inspiration followed by rapid movement of the limbs. This is the physiological and pharmacologically validated basis for the 4-s exercise test (4sET) used to assess the vagal modulation of cardiac chronotropism. Objective: To present reference data for 4sET in healthy adults. Methods: After applying strict clinical inclusion/exclusion criteria, 1,605 healthy adults (61% men) aged between 18 and 81 years subjected to 4sET were evaluated between 1994 and 2014. Using 4sET, the cardiac vagal index (CVI) was obtained by calculating the ratio between the duration of two RR intervals in the electrocardiogram: 1) after a 4-s rapid and deep breath and immediately before pedaling and 2) at the end of a rapid and resistance-free 4-s pedaling exercise. Results: CVI varied inversely with age (r = -0.33, p < 0.01), and the intercepts and slopes of the linear regressions between CVI and age were similar for men and women (p > 0.05). Considering the heteroscedasticity and the asymmetry of the distribution of the CVI values according to age, we chose to express the reference values in percentiles for eight age groups (years): 18–30, 31–40, 41–45, 46–50, 51–55, 56–60, 61–65, and 66+, obtaining progressively lower median CVI values ranging from 1.63 to 1.24. Conclusion: The availability of CVI percentiles for different age groups should promote the clinical use of 4sET, which is a simple and safe procedure for the evaluation of vagal modulation of cardiac chronotropism.

Comparasion of polyacrylamide gel electrophoresis (PAGE), immuno-electron microscopy (IEM) and enzyme immunoassay (EIA) for the rapid diagnosis of rotavirus infection in children

Detection of rotavirus RNA by polyacrylamide gel electrophoresis (PAGE) proved to be a highly sensitive and rapid diagnostic test. A comparison of this assay with immuno-electron microscopy (IEM) and enzyme immunoassay (EIA) in 245 faeces from children with gastroenteritis revealed complete agreement between the three assays in 238 (97.14%) samples. Among 75 samples positive in at least one of the three assays, negative results were observed in 5 (6.48%) by PAGE, in 6 (6.76%) by EIA and in none by IEM. Silver staining greatly increased the sensitivity of the PAGE assay. We conclude that although IEM remains the most sensitive and rapid rotavirus diagnostic assay, the PAGE technique has many advantages in its favour, including the non-requirement of expensive equipment, the use of only chemically defined reagents and the capacity to distinguish virus subgroup and variants and to detect non-crossreactive rotaviruses which are missed in serological assays.

Dispersal of phlebotomine sand flies (Diptera: Psychodidae) in a colombian focus of leishmania (Viannia) brasiliensis

Dispersal of five species of phlebotomine sand flies was studied in a coffee plantation near Arboledas, Colombia by mark-release-recapture studies using fluorescent powders. The estimated recapture rate for males of Lutzomyia shannoni marked and released during the day was 28.1% significantly higher than that for all other species (p < 0.05). Recapture rate of female Lu. shannoni was 9.5% and no females of the other four species were recovered. This suggests either that Lu. shannoni is a more sedentary species than the others, or that the large trees on wich these insects were captured and recaptured function as foci of lekking behaviour in males. The high recapture rates of females of this species may indicate that oviposition occurs in close proximity to the bases of these trees. Although most marked sand flies were recaptured within 200 m of their release point, a single female Lu. gomezi was recovered 960 m away 36 h after release. This suggests that the dispersal capacity of Lutzomyia species may be greater than has been though, an important consideration in future control programs directed against these insects in Leishmania-endemic areas.

A rapid, reliable method of evaluating growth and viability of intraerythrocytic protozoan hemoparasites using fluorescence flow cytometry

Fluorescence flow cytometry was employed to assess the potential of a vital dye, hydroethiedine, for use in the detection and monitoring of the viability of hemoparasites in infected erythrocytes, using Babesia bovis as a model parasite. The studies demonstrated that hydroethidine is taken up by B. bovis and metabolically converted to the DNA binding fluorochrone, ethidium. Following uptake of the dye, erythrocytes contamine viable parasites were readily distinguished and quantitated. Timed studies with the parasiticidal drug, Ganaseg, showed that it is possible to use the fluorochrome assay to monitor the effects of the drug on the rate of replication and viability of B. bovis in culture. The assay provides a rapid method for evaluation of the in vitro effect of drugs on hemoparasites and for analysis of the effect of various components of the immune response, such as lymphokines, monocyte products, antibodies, and effector cells (T, NK, LAK, ADCC) on the growth and viability of intraerythrocytic parasites.

An inhibition ELISA to determine alpha macroglobulin levels in mouse plasma

A sensitive method for quantifying mouse plasma alpha-macroglobulins (AM) using an inhibition ELISA is described. AM are important plasmaproteinase inhibitors that possibly act also as immunomodulatory molecules. The standard protocol develope in our experiments involves coating well with 10 µg/ml A2M in carbonate buffer, followed by incubation with a 1:1 (v/v) mixture of the plasma to be tested (diluted 1/1000) and goat anti-AM (diluted 1/1250). This is followed by further incubation, first with the enzyme-conjugated antibody and with the substrate prior to the reading of absorbance levels of the reaction products. Standard curve samples must be included in each plate, employing known amounts of the purified Murine Alpha-2-Macroglobulin (MuA2M) used for coating, with concentrations ranging from 0.001 to 10 µg/ml. Using test samples in triplicates and a 6-point standard curve in a single ELISA plate, 25 plasma samples can be tested accurately. The method offers an useful tool for establishing AM levelsin small samples of mouse plasma.

Diagnosis of malaria by acridine orange fluorescent microscopy in an endemic area of Venezuela

Fluorescent (acridine orange) microscopical examination of capillary centrifuged blood (quantitative buffy coat [QBC®] analysis) and Giemsa stained thick blood smears (GTS) were compared for diagnosis of malaria in blood specimens from adults living in malaria transmission areas of the States of Bolivar and Amazonas in southeastern and south Venezuela, respectively. Of a total of 198 GTS examined, 95 subjects (48%) showed parasitaemia. Among the 95 blood films with a positive GTS, 94 were judged positive by the QBC. However, positive QBC tubes were found in 29 out of 103 blood specimens with a negative GTS. Thus, relative to a GTS standard, the sensitivity and specificity of the QBC-test was 99.2% and 72%, respectively. Young trophozoites of Plasmodium vivax and P. falciparum could not be distinguished with certainty. It is confirmed that the QBC offers many advantages compared with the standard diagnosis of malaria parasites, specifically in the speed of staining and ease of interpretation. However, in places where P. falciparum and P. vivax occur, species and stage differentiation should be confirmed with the GTS.

Response to Heterologous Leishmanins in Cutaneous Leishmaniasis in Nigeria: Discovery of a New Focus

A pilot study was undertaken to preliminary illustrate the leishmanin skin test (LST) positivity to distinct antigen preparations (derived from promastigote of either Leishmania major or L. amazonensis, or pooled L. mexicana, L. amazonensis and L. guyanensis) in cutaneous leishmaniasis (CL) patients and healthy subjects living in two endemic foci in Nigeria. The study was designed to provide insights into whether cross-species leishmanin, such as that prepared from New World Leishmania could be useful to detect cases of Old World leishmanial infection and to compare the results with LST using L. major-derived leishmanin. The overall LST positivity in individuals from Keana tested with the cross-species leishmanin was 28.7% (27/94), while the positivity rate in the subjects from Kanana tested with the same leishmanin was 54.5% (6/11). Lower positivity values were obtained when L. major (12.5%; 11/88) or L. amazonensis (15.8%; 9/57) was tested as antigen in grossly comparable populations. Moreover, the pooled leishmanin identified most of the subjects (13/14; 92.9%) with active or healed CL, and the maximum reaction sizes were found among positive subjects in this group. No healthy controls (10 total) showed specific DTH response. The LST was useful for assessing the prevalence of subclinical infection and for measuring CL transmission over time. We report for the first time the occurrence of CL in Kanana village of Langtang South local government area of Plateau State

In vitro Comparison of Disk Diffusion and Agar Dilution Antibiotic Susceptibility Test Methods for Neisseria gonorrhoeae

At present, most Neisseria gonorrhoeae testing is done with ß-lactamase and agar dilution tests with common therapeutic agents. Generally, in bacteriological diagnosis laboratories in Argentina, study of antibiotic susceptibility of N.gonorrhoeae is based on ß-lactamase determination and agar dilution method with common therapeutic agents. The National Committee for Clinical Laboratory Standards (NCCLS) has recently described a disk diffusion test that produces results comparable to the reference agar dilution method for antibiotic susceptibility of N.gonorrhoeae, using a dispersion diagram for analyzing the correlation between both techniques. We obtained 57 gonococcal isolates from patients attending a clinic for sexually transmitted diseases in Tucumán, Argentina. Antibiotic susceptibility tests using agar dilution and disk diffusion techniques were compared. The established NCCLS interpretive criteria for both susceptibility methods appeared to be applicable to domestic gonococcal strains. The correlation between the MIC's and the zones of inhibition was studied for penicillin, ampicillin, cefoxitin, spectinomycin, cefotaxime, cephaloridine, cephalexin, tetracycline, norfloxacin and kanamycin. Dispersion diagrams showed a high correlation between both methods.

Comparison between the Counter Immunoelectrophoresis Test and Mouse Neutralization Test for the Detection of Antibodies against Rabies Virus in Dog Sera

The detection of rabies antibodies is extremely valuable for epidemiological studies, determination of immune status in man, animals, and for the diagnosis of the disease. Several serological procedures have been described for this purpose. The present study reports a comparison between counterimmunoelectrophoresis test (CIET) and mouse neutralization test (MNT) in the detection of antibodies against rabies virus from 212 serum samples of vaccinated dogs. The agreement between both techniques was 79.7% and a significative association was demonstrated. The correlation coefficients between MNT and the CIET titers was determined considering 88 samples showing positive results in both techniques [CIET = 2 and MNT = 5 (0.13 IU/ml)] and resulted r² = 0.7926 (p < 0.001). The performance of CIET system was technically simple, cheap and rapid, and thereby it could be useful for serological monitoring of dog vaccination campaigns as well as for individual analysis.

Direct Sensitivity Test of the MB/BacT System

In order to evaluate the direct-method test of sensitivity to drugs used in the principal tuberculosis treatment regimes, in the Organon Teknika MB/BacT system, we tested 50 sputum samples positive to microscopy taken from patients with pulmonary tuberculosis and with clinical indications for an antibiogram, admitted sequentially for examination during the routine of the reference laboratory. The material was treated v/v with 23% trisodium phosphate solution, incubated for 24 h at 35°C, and neutralized v/v with 20% monosodium phosphate solution. The material was then centrifuged and the sediment inoculated into flasks containing Rifampin - 2 µg/ml, Isoniazid - 0.2 µg/ml, Pyrazinamide - 100 µg/ml, Ethambutol - 2.5 µg/ml, Ethionamide - 1.25 µg/ml, and Streptomycin - 2 µg/ml. The tests were evaluated using the indirect method in the BACTEC 460 TB (Becton Dickinson) system as the gold standard. The results showed that the Rifampin test performed best, i.e., 100% sensitivity at 95% Confidence Interval (82.2-100) and 100% specificity at 95% Confidence Interval (84.5-100), followed by Isoniazid and Pyrazinamide. In this experiment, 92% of the materials showed a final reading in 30 days; this period represents the time for primary isolation as well as the results of the sensitivity profile, and is within Centers for Disease Control and Prevention recommendations regarding time for performance of the antibiogram. The inoculated flasks showed no contamination during the experiment. The MB/BacT is shown to be a reliable, rapid, fully automated nonradiometric system for the tuberculosis antibiogram.

Comparison of a Recombinant-antigen Enzyme Immunoassay with Treponema pallidum Hemagglutination Test for Serological Confirmation of Syphilis

A recombinant-antigen enzyme immunoassay (EIA), BioSCREEN TM anti-Treponema pallidum, was compared favorably with the T. pallidum hemagglutination test, in the detection of specific antibodies in different groups of sera from patients with primary (n = 38), secondary (n = 10), early latent (n = 28) and congenital syphilis (n = 2), patients with leptospirosis ( n= 8), infectious mononucleosis (n = 7), hepatitis (n = 9), diabetes mellitus (n = 11), rheumatoid arthritis (n = 13), leprosy (n = 11), tuberculosis (n = 9), HIV/Aids ( n= 12), systemic lupus erythematosus (n = 4), rheumatic fever (n = 3), old-persons (n = 9), pregnant women (n = 29) and blood donors (n = 164). The coincidence between them was 95.1%. The sensitivity and specificity of the EIA were 93.3% and 95.5%, respectively. Fifteen serum specimens belonging to old-persons, pregnant women, blood donors, and patients with human leptospirosis, hepatitis, diabetes mellitus, tuberculosis and rheumatic fever gave false-positive results by Venereal Disease Research Laboratory and/or Rapid Plasma Reagin. The EIA can be used as alternative method for the serological confirmation of syphilis.

IgM-Immunofluorescence Test as a Diagnostic Tool for Epidemiologic Studies of Schistosomiasis in Low Endemic Areas

The high sensitivity and the ability to diagnose schistosomiasis in a very early phase after infection have indicated the detection of IgM antibodies to Schistosoma mansoni gut antigens by the immunofluorescence test (IgM-IFT) as a useful serological test for epidemiological studies in low endemic areas. When applied in a follow-up study for two years, higher rates of seroconversion from IFT negative to positive were observed during the summer months, suggesting seasonal transmission of schistosomiasis in the rural area of the municipality of Itariri (São Paulo, Brazil). In each survey, blood samples from about 600 schoolchildren were collected on filter paper and submitted to IgM-IFT. When the blood samples were classified for the IgM antibody levels, according to the intensity of fluorescent reaction observed at fluorescence microscopy, and correlated to the egg counts in the Kato-Katz positive patients, no association was observed. This observation might suggest that the intensity of fluorescence observed in the IgM-IFT, as an indicator of IgM antibody levels, could not be an useful seroepidemiological marker for classifying areas of low endemicity according to degrees of infection.

Evaluation of the direct agglutination test and the rK39 dipstick test for the sero-diagnosis of visceral leishmaniasis

The direct agglutination test (DAT) based on a freeze-dried antigen and the rK39 dipstick test were evaluated for the sero-diagnosis of visceral leishmaniasis (VL). The sensitivity and specificity of both tests were determined using sera from confirmed VL patients (n = 21), healthy controls (n = 19) and from patients with other confirmed infectious diseases (n = 42). The DAT had a sensitivity and a specificity of 100%. The rK39 had a sensitivity of 85.7% and a specificity of 82%. Both tests were also used to screen blood samples of confirmed VL patients (n = 15) and serum samples of VL suspects (n = 61). The DAT found all blood samples of confirmed VL patients positive and tested 98.4% of the serum samples of the VL suspects positive. In contrast, rK39 detected in 9/15 blood samples (60%) antibodies against Leishmania chagasi and found 85.3% of the serum samples of the suspected patients positive. Although the rK39 dipstick is more rapid and user friendlier than the DAT, the latter has a superior sensitivity and specificity. Furthermore, the reagents used for DAT do not require cold storage, whereas the buffer of the rK39 must be stored at 4ºC. Therefore, the DAT is the most suitable test for the sero-diagnosis of VL under field conditions.