HIGH SENSITIVE PCR METHOD FOR DETECTION OF PATHOGENIC Leptospira spp. IN PARAFFIN-EMBEDDED TISSUES

This study describes the development and application of a new PCR assay for the specific detection of pathogenic leptospires and its comparison with a previously reported PCR protocol. New primers were designed for PCR optimization and evaluation in artificially-infected paraffin-embedded tissues. PCR was then applied to post-mortem, paraffin-embedded samples, followed by amplicon sequencing. The PCR was more efficient than the reported protocol, allowing the amplification of expected DNA fragment from the artificially infected samples and from 44% of the post-mortem samples. The sequences of PCR amplicons from different patients showed >99% homology with pathogenic leptospires DNA sequences. The applicability of a highly sensitive and specific tool to screen histological specimens for the detection of pathogenic Leptospira spp. would facilitate a better assessment of the prevalence and epidemiology of leptospirosis, which constitutes a health problem in many countries.

In-house ELISA method to analyze anti-Trypanosoma cruzi IgG reactivity for differential diagnosis and evaluation of Chagas disease morbidity

INTRODUCTION: The goal was to develop an in-house serological method with high specificity and sensitivity for diagnosis and monitoring of Chagas disease morbidity. METHODS: With this purpose, the reactivities of anti-T. cruzi IgG and subclasses were tested in successive serum dilutions of patients from Berilo municipality, Jequitinhonha Valley, Minas Gerais, Brazil. The performance of the in-house ELISA was also evaluated in samples from other relevant infectious diseases, including HIV, hepatitis C (HCV), syphilis (SYP), visceral leishmaniasis (VL), and American tegumentary leishmaniasis (ATL), and noninfected controls (NI). Further analysis was performed to evaluate the applicability of this in-house methodology for monitoring Chagas disease morbidity into three groups of patients: indeterminate (IND), cardiac (CARD), and digestive/mixed (DIG/Mix), based on their clinical status. RESULTS: The analysis of total IgG reactivity at serum dilution 1:40 was an excellent approach to Chagas disease diagnosis (100% sensitivity and specificity). The analysis of IgG subclasses showed cross-reactivity, mainly with NI, VL, and ATL, at all selected serum dilutions. Based on the data analysis, the IND group displayed higher IgG3 levels and the DIG/Mix group presented higher levels of total IgG as compared with the IND and CARD groups. CONCLUSIONS: These findings demonstrated that methodology presents promising applicability in the analysis of anti-T. cruzi IgG reactivity for the differential diagnosis and evaluation of Chagas disease morbidity.

Parasitological stool sample exam by spontaneous sedimentation method using conical tubes: effectiveness, practice, and biosafety

INTRODUCTION: Spontaneous sedimentation is an important procedure for stool examination. A modification of this technique using conical tubes was performed and evaluated. METHODS: Fifty fecal samples were processed in sedimentation glass and in polypropylene conical tubes. Another 50 samples were used for quantitative evaluation of protozoan cysts. RESULTS: Although no significant differences occurred in the frequency of protozoa and helminths detected, significant differences in protozoan cyst counts did occur. CONCLUSIONS: The use of tube predicts a shorter path in the sedimentation of the sample, increases concentration of parasites for microscopy analysis, minimizes the risks of contamination, reduces the odor, and optimizes the workspace.

A rapid and simple method to detect ESBL in Enterobacter cloacae based on MIC of cefepime

INTRODUCTION: The aim of this study was to identify a rapid and simple phenotypic method for extended-spectrum β-lactamase (ESBL) detection in Enterobacter cloacae. METHODS: A total of 79 consecutive, non-repeated samples of E. cloacae were evaluated. Four phenotypic methods were applied for ESBL detection, results were compared to multiplex polymerase chain reaction (PCR) as the gold standard reference method: 1) ceftazidime and cefotaxime disks with and without clavulanate, both with boronic acid added; 2) disk approximation using cefepime and amoxicillin/clavulanate; 3) ESBL screening by minimum inhibitory concentration (MIC) ≥ 16µg/mL and 4) by MIC ≥ 2µg/mL for cefepime. RESULTS: Method 4 showed the best combination of sensitivity (100%) and specificity (94%). CONCLUSIONS: MIC ≥ 2µg/mL for cefepime would be very useful for the phenotypic detection of ESBL in samples of E. cloacae.

Polymerase chain reaction-based method for the identification of Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in mucosal tissues conserved in paraffin

ABSTRACTINTRODUCTION: In the Americas, mucosal leishmaniasis is primarily associated with infection by Leishmania (Viannia) braziliensis. However, Leishmania (Viannia) guyanensis is another important cause of this disease in the Brazilian Amazon. In this study, we aimed at detecting Leishmaniadeoxyribonucleic acid (DNA) within paraffin-embedded fragments of mucosal tissues, and characterizing the infecting parasite species.METHODS: We evaluated samples collected from 114 patients treated at a reference center in the Brazilian Amazon by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses.RESULTS: Direct examination of biopsy imprints detected parasites in 10 of the 114 samples, while evaluation of hematoxylin and eosin-stained slides detected amastigotes in an additional 17 samples. Meanwhile, 31/114 samples (27.2%) were positive for Leishmania spp. kinetoplast deoxyribonucleic acid (kDNA) by PCR analysis. Of these, 17 (54.8%) yielded amplification of the mini-exon PCR target, thereby allowing for PCR-RFLP-based identification. Six of the samples were identified as L. (V.) braziliensis, while the remaining 11 were identified as L. (V.) guyanensis.CONCLUSIONS: The results of this study demonstrate the feasibility of applying molecular techniques for the diagnosis of human parasites within paraffin-embedded tissues. Moreover, our findings confirm that L. (V.) guyanensisis a relevant causative agent of mucosal leishmaniasis in the Brazilian Amazon.

The use of a modular titanium endoprosthesis in skeletal reconstructions after bone tumor resections: method presentation and analysis of 37 cases

We analyzed 37 patients who underwent segmental wide resection of bone tumors and reconstruction with a modular titanium endoprosthesis at the Orthopaedic Oncology Group, between 1992 and 1998. Twelve patients were male and 25 were female, with a mean age of 30 years (9 - 81). The mean follow-up was 14 months (2 - 48). The diagnoses were: osteosarcoma (14 cases), metastatic carcinoma (10), Ewing's sarcoma (4), giant cell tumor (4), malignant fibrous histiocytoma (3), chondrosarcoma (1), and aneurysmal bone cyst (1). Eleven articulated total knee, 8 partial proximal femur with bipolar acetabulum, 8 partial proximal humerus, 3 total femur, 2 partial proximal tibia, 2 diaphyseal femur, 2 diaphyseal humerus, and 1 total proximal femur with cementless acetabulum endoprosthesis implant procedures were done. The complications related to the procedure included: infection (5 cases), dislocation (3), module loosening (1), and ulnar nerve paresthesia (1). We used the following criteria for the clinical evaluation: presence of pain, range of motion, reconstruction stability, surgical and oncologic complications, and patient acceptance. The results were good in 56.8% of the cases, regular in 32.4% and poor in 10.8%.

New method for evaluation of cutaneous sensibility in diabetic feet: preliminary report

Diabetic neuropathy is an important complication of the disease, responsible for ulceration and amputation of the foot. Prevention of these problems is difficult mainly because there is no method to correctly access sensibility on the skin of the foot. The introduction of the Pressure-Specified Sensory Device (PSSD TM) in the last decade made possible the measurement of pressure thresholds sensed by the patient, such as touch, both static and in movement, on a continuous scale. This paper is the first in Brazil to report the use of this device to measure cutaneous sensibility in 3 areas of the foot: the hallux pulp, the calcaneus, and the dorsum, which are territories of the tibial and fibular nerves. METHOD: Non-diabetic patients were measured as controls, and 2 groups of diabetic patients - with and without ulcers - were compared. The PSSD TM was used to test the 3 areas described above. The following were evaluated: 1 PS (1-point static), 1 PD (1-point dynamic), 2 PS (2-points static), 2 PD (2-points dynamic). RESULTS: The diabetic group had poorer sensibility compared to controls and diabetics with ulcers had poorer sensibility when compared to diabetics without ulcers. The differences were statistically significant (P <.001). CONCLUSION: Due to the small number of patients compared, the results should be taken as a preliminary report.

PKO: alternative method for isolating mycobacteria from sputum

We elaborated an alternative culture method, which we denominated PKO (initials in tribute of respect to Petroff, Kudoh and Ogawa), for isolating Mycobacterium tuberculosis from sputum for diagnosis of pulmonary tuberculosis (TB), and to compare its performance with the Swab and Petroff methods. For the technique validation, sputum samples from patients suspected of pulmonary TB cases were examined by acid-fast microscopy (direct and concentrated smear), PKO, Swab and Petroff methods. We found that Petroff and PKO methods have parity in the effectiveness of M. tuberculosis isolation. However, by the PKO method, 65% of isolated strains were detected in a period of £15 days, while by the Petroff method the best detection was in an interval of 16-29 days (71%). In positive smear samples, the average time of PKO isolation is only superior to the one related for Bactec 460TB. In conclusion, the exclusion of the neutralization stage of pH in the PKO reduces the manipulation of the samples, diminishes the execution time of the culture according to the Petroff method and facilitates the qualification of professionals involved in the laboratorial diagnosis of Tuberculosis.

Comorbidities of mental and behavioral disorders in chemically dependent patients in different periods of abstinence

OBJECTIVE: To assess the frequency of comorbidities of mental and behavioral disorders (CMBD) in psychoactive substance (PAS)-dependent patients with different periods of abstinence cared for at Alcohol and Other Drug Psychosocial Care Centers (CAPS-ad). METHOD: All patients under treatment in the two CAPS-ad of the city of Uberlândia-MG, between April and September 2010, were consecutively assessed. The ICD-10 symptom checklist was used to diagnose CMBD; additional information was obtained from interviews and medical records. The patients were divided according to duration of abstinence: < 1 week (Group 1); 1-4 weeks (Group 2); and > 4 weeks (Group 3). RESULTS: Of all patients assessed, 62.8% were diagnosed with CMBD, which were more frequent (p < 0.05) in Group 1 (72%) than Group 3 (54.2%), and both groups were similar to Group 2 (61%). Depressive and anxiety disorders were more frequent among patients of Group 1. Mood disorders were more frequent (p < 0.05) in women [22/34 (65%) vs. 54/154 (35.1%)], whereas psychotic disorders were more frequent (p = 0.05) in men [16/154 (10.4%) vs. 0]. The presence of CMBD was associated with more severe clinical conditions. CONCLUSIONS: The higher frequency of diagnosis of CMBD in patients of Group 1 may have resulted from the difficulties in distinguishing mental disorders that are due to PAS intoxication or withdrawal from those that are not. However, to make the diagnosis of CMBD, even during detoxification, can increase the likelihood of better response to treatment.

Cognitive processes affect the gait of subjects with Parkinson’s and Alzheimer’s disease in dual tasks

Objective To investigate the relation between gait parameters and cognitive impairments in subjects with Parkinson’s disease (PD) and Alzheimer’s disease (AD) during the performance of dual tasks. Methods This was a cross-sectional study involving 126 subjects divided into three groups: Parkinson group (n = 43), Alzheimer group (n = 38), and control group (n = 45). The subjects were evaluated using the Timed Up and Go test administered with motor and cognitive distracters. Gait analyses consisted of cadence and speed measurements, with cognitive functions being assessed by the Brief Cognitive Screening Battery and the Clock Drawing Test. Statistical procedures included mixed-design analyses of variance to observe the gait patterns between groups and tasks and the linear regression model to investigate the influence of cognitive functions in this process. A 5% significant level was adopted. Results Regarding the subjects’ speed, the data show a significant difference between group vs task interaction (p = 0.009), with worse performance of subjects with PD in motor dual task and of subjects with AD in cognitive dual task. With respect to cadence, no statistical differences was seen between group vs task interaction (p = 0.105), showing low interference of the clinical conditions on such parameter. The linear regression model showed that up to 45.79%, of the variance in gait can be explained by the interference of cognitive processes. Conclusion Dual task activities affect gait pattern in subjects with PD and AD. Differences between groups reflect peculiarities of each disease and show a direct interference of cognitive processes on complex tasks.

Presence of dual diagnosis between users and non-users of licit and illicit drugs in Brazil

ABSTRACT Objective Investigate the occurrence of dual diagnosis in users of legal and illegal drugs. Methods It is an analytical, cross-sectional study with a quantitative approach, non-probabilistic intentional sampling, carried out in two centers for drug addiction treatment, by means of individual interviews. A sociodemographic questionnaire, the Alcohol, Smoking and Substance Involvement Screening Test (ASSIST) and the Mini-International Neuropsychiatric Interview (MINI) were used. Results One hundred and ten volunteers divided into abstinent users (group 1), alcoholics (group 2) and users of alcohol and illicit drugs (group 3). The substances were alcohol, tobacco, crack and marijuana. A higher presence of dual diagnosis in group 3 (71.8%) was observed, which decreased in group 2 (60%) and 37.1% of drug abstinent users had psychiatric disorder. Dual diagnosis was associated with the risk of suicide, suicide attempts and the practice of infractions. The crack consumption was associated with the occurrence of major depressive episode and antisocial personality disorder. Conclusion It was concluded that the illicit drug users had a higher presence of dual diagnosis showing the severity of this clinical condition. It is considered essential that this clinical reality is included in intervention strategies in order to decrease the negative effects of consumption of these substances and provide better quality of life for these people.

Reliability of the interpretation of coronary angiography by the simple visual method

OBJECTIVE: Evaluation of inter and intraobserver reproducibility of by the visual method interpretation of cineangiogram in a clinically based context. METHODS: Five interventional cardiologists analyzed 11 segments of 8 coronary cineangiograms at a two month apart sessions. The percent luminal reduction by the lesions were analyzed by two different classifications: in one (A) the lesions were graded in 0% = absent, 1-50% = mild, 51 - 69 = moderate, and > or = 70% = severe; the other classification (B) was a dichotomic one : <70% = nonsignificant and > or = 70%=significant lesions. The agreement were measured by the kappa (k) index. RESULTS: Interobserver agreement was moderate for classification A (1st measurement, k = 0.36 -- 0.63, k m = 0.49; 2nd measurement, k = 0.39-0.68, k m = 0.52) and good for classification B (1st measurement, k = 0.55-0.73, k m = 0.63; 2nd measurement, k = 0.37-0.82, k m = 0.61). Intraobserver levels of agreement were k = 0.57-0.95 for classification A and 0.62-1.0 for classification B. CONCLUSION: The higher level of reproducibility obtained by adopting the dichotomous criteria usually considered for ischemic limits demonstrates that in the present clinical context, the reliability of the simple visual method is adequate for the identification of patients with clinically significant lesions and candidates for myocardial revascularization procedures.

Dual anterior descending coronary artery associated with coronary artery disease

The patient is a male with risk factors for coronary artery disease, who was referred for cardiac catheterization after acute myocardial infarction in the inferior wall. The patient underwent transluminal coronary angioplasty in the right coronary artery with successful stent implantation.

Quantitative method of viral pollution determination for large volume of water using ferric hydroxide gel impregnated on the surface of glassfibre cartridge

Quantitative method of viral pollution determination for large volume of water using ferric hydroxide gel impregnated on the surface of glassfibre cartridge filter. The use of ferric hydroxide gel, impregnated on the surface of glassfibre cartridge filter enable us to recover 62.5% of virus (Poliomylitis type I, Lsc strain) exsogeneously added to 400 liters of tap-water. The virus concentrator system consists of four cartridge filters, in which the three first one are clarifiers, where the contaminants are removed physically, without significant virus loss at this stage. The last cartridge filter is impregnated with ferric hydroxide gel, where the virus is adsorbed. After the required volume of water has been processed, the last filter is removed from the system and the viruses are recovered from the gel, using 1 liter of glycine/NaOH buffer, at pH 11. Immediately the eluate is clarified through series of cellulose acetate membranes mounted in a 142mm Millipore filter. For the second step of virus concentration, HC1 1N is added slowly to the eluate to achieve pH 3.5-4. MgC1, is added to give a final concentration of 0.05M and the viruses are readsorbed on a 0.45 , porosity (HA) cellulose acetate membrane, mounted in a 90 mm Millipore filter. The viruses are recovered using the same eluent plus 10% of fetal calf serum, to a final volume of 3 ml. In this way, it was possible to concentrate virus from 400 liters of tap-water, into 1 liter in the first stage of virus concentration and just to 3 ml of final volume in a second step. The efficiency, simplicity and low operational cost, provded by the method, make it feasible to study viral pollution of recreational and tap-water sources.

A simple method to purify biologically and antigenically preserved bloodstream trypomastigotes of Trypanosoma cruzi using Deae-cellulose columns

A method to purify trypanosomastigotes of some strains of Trypanosoma cruzi (Y, CL, FL, F, "Berenice", "Colombiana" and "São Felipe") from mouse blood by using DEAE-cellulose columns was standardized. This procedure is a modification of the Lanham & Godfrey methods and differs in some aspects from others described to purify T. cruzi bloodstream trypomastigotes, mainly by avoidance of prior purifications of parasites. By this method, the broad trypomastigotes were mainly isolated, accounting for higher recoveries obtained with strains having higher percentages of these forms: processing of infected blood from irradiated mice could be advantageous by increasing the recovery of parasites (percentage and/or total number) and elution of more slender trypomastigotes. Trypomastigotes purified by this method presented normal morphology and motility, remained infective to triatomine bugs and mice, showing in the latter prepatent periods and courses parasitemia similar to those of control parasites, and also reproducing the polymorphism pattern of each strain. Their virulence and pathogenicity also remained considerably preserved, the latter property being evaluated by LD 50 tests, mortality rates and mean survival time of inoculated mice. Moreover, these parasites presented positive, clear and peripheral immunofluorescence reaction at titres similar to those of control organisms, thus suggesting important preservation of their surface antigens.