TESTING A SUBTYPE-SPECIFIC GP41 AMPLIFICATION METHOD FOR GENOTYPING INDIVIDUALS INFECTED BY HUMAN IMMUNODEFICIENCY VIRUS TYPE-1 IN THE BRAZILIAN POPULATION OF ITAJAÍ, SOUTH BRAZIL

The method used by YAGYU et al. for the subtype-specific polymerase chain reaction (PCR) amplification of the gp41 transmembrane region of the human immunodeficiency virus type-1 (HIV-1) env gene, was tested. HIV-1 proviral DNA from 100 infected individuals in Itajaí, South Brazil was used to analyze this method. Seventy individuals were determined according to this method as having PCR products at the expected size for subtypes B, C, D and F. Of these individuals, 26 (37.1%) were observed as having the expected amplification for subtype C, and 42 (60%) were observed as having the expected products for subtypes B and D. Of the subtype B and D amplicons, 16 (22.9%) were classified as subtype D, and 26 (37.1%) were classified as subtype B. Two individuals (2.9%) had amplicons that were observed after subtype F-specific amplification was performed. Sequencing and comparing the patient sequences to reference sequences confirmed the classification of sequences of subtypes C and B. However, sequences that were falsely determined as being D and F in the PCR assay were determined as being subtypes C and B, respectively, by sequence analysis. For those individuals from whom no amplified products were obtained, a low viral load that was indicated in their patient history may explain the difficulty in subtyping by PCR methods. This issue was demonstrated by the results of ANOVA when testing the effect of viral load on the success of PCR amplification. The alignment of the obtained sequences with HIV-1 reference sequences demonstrated that there is high intra-subtype diversity. This indicates that the subtype-specific primer binding sites were not conserved or representative of the subtypes that are observed in the Brazilian populations, and that they did not allow the correct classification of HIV-1 subtypes. Therefore, the proposed method by YAGYU et al. is not applicable for the classification of Brazilian HIV-1 subtypes.

COMPARISON BETWEEN AUTOMATED SYSTEM AND PCR-BASED METHOD FOR IDENTIFICATION AND ANTIMICROBIAL SUSCEPTIBILITY PROFILE OF CLINICAL Enterococcus spp

Enterococci are increasingly responsible for nosocomial infections worldwide. This study was undertaken to compare the identification and susceptibility profile using an automated MicrosScan system, PCR-based assay and disk diffusion assay of Enterococcus spp. We evaluated 30 clinical isolates of Enterococcus spp. Isolates were identified by MicrosScan system and PCR-based assay. The detection of antibiotic resistance genes (vancomycin, gentamicin, tetracycline and erythromycin) was also determined by PCR. Antimicrobial susceptibilities to vancomycin (30 µg), gentamicin (120 µg), tetracycline (30 µg) and erythromycin (15 µg) were tested by the automated system and disk diffusion method, and were interpreted according to the criteria recommended in CLSI guidelines. Concerning Enterococcus identification the general agreement between data obtained by the PCR method and by the automatic system was 90.0% (27/30). For all isolates of E. faecium and E. faecalis we observed 100% agreement. Resistance frequencies were higher in E. faecium than E. faecalis. The resistance rates obtained were higher for erythromycin (86.7%), vancomycin (80.0%), tetracycline (43.35) and gentamicin (33.3%). The correlation between disk diffusion and automation revealed an agreement for the majority of the antibiotics with category agreement rates of > 80%. The PCR-based assay, the van(A) gene was detected in 100% of vancomycin resistant enterococci. This assay is simple to conduct and reliable in the identification of clinically relevant enterococci. The data obtained reinforced the need for an improvement of the automated system to identify some enterococci.

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.

Impact of the antipneumococcal conjugate vaccine on the occurrence of infectious respiratory diseases and hospitalization rates in children

INTRODUCTION: In 2010, to reduce the occurrence of serious pneumococcal disease, the Ministry of Health in Brazil incorporated the 10-valent pneumococcal vaccine in the immunization schedule of children younger than two years of age. The objective of this study was to evaluate the impact of vaccination on the incidence of infectious respiratory diseases in infants before and after the introduction of the 10-valent pneumococcal vaccine. METHODS: This cross-sectional study involved primary care and hospital networks from a city in Minas Gerais State, Brazil, between 2009 and 2012. RESULTS: A 40% reduction in the prevalence of community-acquired pneumonia (CAP) was observed after introducing the pneumococcal conjugate vaccine. Male children were 28% more likely to develop the disease. The prevalence ratio ([PR] = 1.96, 95% CI: 1.52 to 2.53, p < 0.05) suggested that not being vaccinated was associated with the occurrence of pneumonia. The prevalence of CAP was 70% lower (PR 0.30, 95% CI: 0.24 to 0.37, p<0.05) in children vaccinated as recommended compared to children with delayed vaccination, suggesting that the updated vaccine schedule improves protection. CONCLUSIONS: Immunization with the 10-valent pneumococcal vaccine appeared to reduce the number of pneumonia cases in children during the study period. Prospective studies are needed to confirm the efficacy of the vaccine against the occurrence of pneumococcal pneumonia.

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.

Seasonal and spatial variations in limnological conditions of a floodplain lake (lake Catalão) connected to both the Solimões and Negro Rivers, Central Amazonia

Lakes play an important role in biogeochemical, ecological and hydrological processes in the river-floodplain system. The aim of this study was to evaluate the dynamics of the limnological conditions of Catalão Lake, an Amazon floodplain lake. Thus, some of the main limnological environment variables (O2, temperature, pH, nutrient, electrical conductivity) of the Catalão Lake were analyzed under temporal and spacial scales. The study was conducted between November/2004 and August/2005. Sampling excursion were carried out every three months; one excursion for each of the four different hydrological periods (low water, rising water, high water and falling water). Sampling points were chosen so that it could be obtained a gradient of the distance from Negro River. Limnological profiles in Catalão Lake showed generally acidic to slightly alcaline water, with low levels of dissolved oxygen and low concentrations of soluble reactive phosphorous. The Negro River seems to exert the main influence during the rising water period, while the Solimões River is the principal controlling river during peak water. The Principal Component Analysis (PCA) grouped the seasonal collections by hydrological period, showing the formation of a north-south spatial gradient within the lake in relation to the limnological variables. Multivariate dispersion analysis based on distance-to-centroid method demonstrated an increase in similarity over the course of the hydrological cycle, as the lake was inundated in response to the flood pulse of the main river channels. However, the largest spatial homogeneity in the lake was observed in the epilimnion layer, during the falling water period. The daily analysis of variation indicated an oligomitic pattern during the years in which the lake was permanently connected to the Negro River. Although Catalão Lake receives large quantities of both black water from the Negro River and sediment-filled water from the Solimões River, the physical and chemical characteristics of the lake are more similar to those of the Solimões (várzea lake) than the Negro (blackwater lake).

Palm community transitions along a topographic gradient from floodplain to terra firme in the eastern Amazon

Palms show clear niche segregation patterns along topographic gradients in tropical forests, with some species associated to terra firme and others to seasonally flooded areas. The aim of this study was to quantitatively describe the fine-scale spatial variation within a palm community, tracking the changes in species' abundance along environmental gradients associated with a perennial stream the eastern Amazon. The study of palm communities was based on 60 forest plots in which all adult palms were counted. We found a total of 566 palms in a community containing 11 species. Furthermore, we found a significant separation in the palm community between seasonally-flooded and terra firme forests. We found a gradient with various densities of the three most abundant palm species within the first 100 m away from the flooded area. Other species were located exclusively in the terra firme forest. The abundance of the six most common species were distributed in relation to humidity gradients from floodplains to terra firme, with palm distribution from the most flood-tolerant to the least flood-tolerant palm species as follows: Euterpe oleracea, Attalea phalerata and Socratea exorrhiza (species with floodplain affinity), Astrocaryum gynacanthum, Astrocaryum aculeatum, Attalea maripa (species with terra firme affinity)

Predicting the distribution of forest tree species using topographic variables and vegetation index in eastern Acre, Brazil

Species distribution modeling has relevant implications for the studies of biodiversity, decision making about conservation and knowledge about ecological requirements of the species. The aim of this study was to evaluate if the use of forest inventories can improve the estimation of occurrence probability, identify the limits of the potential distribution and habitat preference of a group of timber tree species. The environmental predictor variables were: elevation, slope, aspect, normalized difference vegetation index (NDVI) and height above the nearest drainage (HAND). To estimate the distribution of species we used the maximum entropy method (Maxent). In comparison with a random distribution, using topographic variables and vegetation index as features, the Maxent method predicted with an average accuracy of 86% the geographical distribution of studied species. The altitude and NDVI were the most important variables. There were limitations to the interpolation of the models for non-sampled locations and that are outside of the elevation gradient associated with the occurrence data in approximately 7% of the basin area. Ceiba pentandra (samaúma), Castilla ulei (caucho) and Hura crepitans (assacu) is more likely to occur in nearby water course areas. Clarisia racemosa (guariúba), Amburana acreana (cerejeira), Aspidosperma macrocarpon (pereiro), Apuleia leiocarpa (cumaru cetim), Aspidosperma parvifolium (amarelão) and Astronium lecointei (aroeira) can also occur in upland forest and well drained soils. This modeling approach has potential for application on other tropical species still less studied, especially those that are under pressure from logging.

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.