Comparison of conventional serology and PCR methods for the routine diagnosis of Trypanosoma cruzi infection

Introduction Trypanosoma cruzi, a flagellated protozoan, is the etiologic agent of Chagas disease, and it is estimated that approximately 5 million people in Brazil are infected with this parasite. This work aimed to compare the current diagnostic methods for Chagas disease, including conventional serological (IFAT and ELISA) and molecular techniques (PCR), to introduce PCR as an auxiliary technique. Methods A total of 106 chagasic patients were evaluated: 88 from endemic areas of Parana, 6 from São Paulo, 3 from Minas Gerais, 3 from Rio Grande do Sul, 1 from Bahia and 5 from the Santa Catarina T. cruzi outbreak. The samples were analyzed by conventional serological methods (IFAT, ELISA), hemoculture and PCR to confirm Chagas disease. Results When IFAT was used to determine antibody levels, the sensitivity was 81.7% for patients with the cardiac form of the disease and 100% for the other clinical forms. In contrast, ELISA showed 84% sensitivity and 100% specificity. The use of serological and molecular techniques and their implications for the diagnosis of Chagas disease in non-endemics area are discussed. Conclusions PCR constitutes an excellent support methodology for the laboratory diagnosis of Chagas disease due to its high sensitivity and specificity.

Bloodstream infection in patients with end-stage renal disease in a teaching hospital in central-western Brazil

Introduction Vascular access in patients undergoing hemodialysis is considered a critical determinant of bloodstream infection (BSI) and is associated with high morbidity and mortality. The purpose of this study was to investigate the occurrence of BSI in patients with end-stage renal disease using central venous catheters for hemodialysis. Methods A cohort study was conducted in a public teaching hospital in central-western Brazil from April 2010 to December 2011. For every patient, we noted the presence of hyperemia/exudation upon catheter insertion, as well as fever, shivering, and chills during hemodialysis. Results Fifty-nine patients were evaluated. Thirty-five (59.3%) patients started dialysis due to urgency, 37 (62.7%) had BSI, and 12 (20%) died. Hyperemia at the catheter insertion site (64.9%) was a significant clinical manifestation in patients with BSI. Statistical analysis revealed 1.7 times more cases of BSI in patients with hypoalbuminemia compared with patients with normal albumin levels. The principal infective agents identified in blood cultures and catheter-tip cultures were Staphylococcus species (24 cases), non-fermentative Gram-negative bacilli (7 cases of Stenotrophomonas maltophilia and 5 cases of Chryseobacterium indologenes), and Candida species (6). Among the Staphylococci identified, 77.7% were methicillin-resistant, coagulase-negative Staphylococci. Of the bacteria isolated, the most resistant were Chryseobacterium indologenes and Acinetobacter baumannii. Conclusions Blood culture was demonstrated to be an important diagnostic test and identified over 50% of positive BSI cases. The high frequency of BSI and the isolation of multiresistant bacteria were disturbing findings. Staphylococcus aureus was the most frequently isolated microorganism, although Gram-negative bacteria predominated overall. These results highlight the importance of infection prevention and control measures in dialysis units.

Evaluation of two sweeping methods for estimating the number of immature Aedes aegypti (Diptera: Culicidae) in large containers

Introduction Here, we evaluated sweeping methods used to estimate the number of immature Aedes aegypti in large containers. Methods III/IV instars and pupae at a 9:1 ratio were placed in three types of containers with, each one with three different water levels. Two sweeping methods were tested: water-surface sweeping and five-sweep netting. The data were analyzed using linear regression. Results The five-sweep netting technique was more suitable for drums and water-tanks, while the water-surface sweeping method provided the best results for swimming pools. Conclusions Both sweeping methods are useful tools in epidemiological surveillance programs for the control of Aedes aegypti.

The performance of four molecular methods for the laboratory diagnosis of congenital toxoplasmosis in amniotic fluid samples

Introduction Toxoplasmosis may be life-threatening in fetuses and in immune-deficient patients. Conventional laboratory diagnosis of toxoplasmosis is based on the presence of IgM and IgG anti-Toxoplasma gondii antibodies; however, molecular techniques have emerged as alternative tools due to their increased sensitivity. The aim of this study was to compare the performance of 4 PCR-based methods for the laboratory diagnosis of toxoplasmosis. One hundred pregnant women who seroconverted during pregnancy were included in the study. The definition of cases was based on a 12-month follow-up of the infants. Methods Amniotic fluid samples were submitted to DNA extraction and amplification by the following 4 Toxoplasma techniques performed with parasite B1 gene primers: conventional PCR, nested-PCR, multiplex-nested-PCR, and real-time PCR. Seven parameters were analyzed, sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), negative likelihood ratio (NLR) and efficiency (Ef). Results Fifty-nine of the 100 infants had toxoplasmosis; 42 (71.2%) had IgM antibodies at birth but were asymptomatic, and the remaining 17 cases had non-detectable IgM antibodies but high IgG antibody titers that were associated with retinochoroiditis in 8 (13.5%) cases, abnormal cranial ultrasound in 5 (8.5%) cases, and signs/symptoms suggestive of infection in 4 (6.8%) cases. The conventional PCR assay detected 50 cases (9 false-negatives), nested-PCR detected 58 cases (1 false-negative and 4 false-positives), multiplex-nested-PCR detected 57 cases (2 false-negatives), and real-time-PCR detected 58 cases (1 false-negative). Conclusions The real-time PCR assay was the best-performing technique based on the parameters of Se (98.3%), Sp (100%), PPV (100%), NPV (97.6%), PLR (∞), NLR (0.017), and Ef (99%).

Incidence of canine leptospirosis in the metropolitan area of Curitiba, State of Parana, Southern Brazil

Introduction The incidence of canine leptospirosis in Brazil needs to be assessed Methods The same dogs in southern Brazil were sampled over two years to determine the prevalence, incidence and association of canine leptospirosis with various risk factors. Results In 2009, the prevalence was 33 (14.4%) of 228 dogs, with a predominance of serovar Canicola (33.4%). In 2010, 90 dogs were re-evaluated (the remaining dogs were lost to deaths, address changes and donations), and the prevalence was found to be 35 (38.9%) of 90, with the predominant serovar being Icterohaemorrhagiae (51.4%). Moreover, the incidence was 26 of 90 (28.9%), and the disease was statistically associated with age (2009) and street access (2010). Conclusions Our findings revealed instability in the dog population and age to be relevant risk factors for canine leptospirosis.

Evaluation of the Chagas Disease Control Program in Açucena Municipality, Rio Doce Valley, State of Minas Gerais, Brazil

Introduction Açucena Municipality, Rio Doce Valley, State of Minas Gerais, Brazil temporarily (2001-2005) interrupted epidemiological surveillance for Chagas disease. The objective of this work was to evaluate the Chagas Disease Control Program (CDCP) in Açucena and to offer suggestions for improving local epidemiological surveillance. Methods This study was conducted in three phases: I) a serological investigation of schoolchildren aged 5 to 15 years using an enzyme-linked immunosorbent assay (ELISA) test performed on blood collected on filter paper followed by ELISA, indirect immunofluorescence (IIF) and indirect hemaglutination (IHA) on venous blood for borderline cases and those in the gray zone of reactivity; II) vector evaluation using the data obtained by local health agents during 2006-2010; and III) examination by ELISA, IIF and IHA of serum samples from the inhabitants of houses where infected Triatoma vitticeps was found and evaluation of their knowledge about Chagas disease. Results Five individuals had inconclusive results in the ELISA screening but were seronegative for Chagas disease. The triatomine evaluation revealed the presence of three species: Triatoma vitticeps, Panstrongylus megistus and Panstrongylus diasi. Triatoma vitticeps was the most prevalent and widespread, with a higher (67%) index of Trypanosoma cruzi flagellates and evidence of colonization. Most of the inhabitants of the infested houses recognized triatomines and had basic knowledge about Chagas disease. Conclusions Although T. vitticeps is not clearly associated with Chagas disease transmission, these results highlight the importance of maintaining CDCP in endemic areas and the need for greater emphasis on epidemiological surveillance, especially in areas with important vectorial changes or that have been modified by human intervention.

Occurrence of potentially pathogenic Vibrio in oysters (Crassostrea gigas) and waters from bivalve mollusk cultivations in the South Bay of Santa Catarina

Introduction This research aimed to identify and quantify potentially pathogenic Vibrio from different cultivations of bivalve shellfish in the State of Santa Catarina, Brazil, and water regions in the South Bay, as well as correlate the incidence of these microorganisms with the physicochemical parameters of marine waters. Methods Between October 2008 and March 2009, 60 oyster and seawater samples were collected from six regions of bivalve mollusk cultivation, and these samples were submitted for Vibrio counts. Results Twenty-nine (48.3%) oyster samples were revealed to be contaminated with one or more Vibrio species. The Vibrio parahaemolyticus and Vibrio vulnificus counts in the samples ranged from < 0.5 log10 Most Probable Number (MPN) g–1 to 2.3 log10 MPN g–1 oyster and from < 0.5 log10 MPN g–1 to 2.1 log10 MPN g–1 oyster, respectively. Of the 60 seawater samples analyzed, 44 (73.3%) showed signs of contamination with one or more vibrio species. The counts of V. parahaemolyticus and V. vulnificus in the samples ranged from < 0.3 log10 MPN·100mL–1 to 1.7 log10MPN·100mL–1 seawater and from < 0.3 log10 MPN·100mL–1 to 2.0 log10 MPN·100mL–1 seawater, respectively. A positive correlation between V. vulnificus counts and the seawater temperature as well as a negative correlation between the V. parahaemolyticus counts and salinity were observed. Conclusions The results suggest the need to implement strategies to prevent vibrio diseases from being transmitted by the consumption of contaminated bivalve shellfish.

The first canine visceral leishmaniasis outbreak in Campinas, State of São Paulo Southeastern Brazil

Introduction Early detection of American visceral leishmaniasis (AVL) outbreak in animals is crucial for controlling this disease in non-endemic areas. Methods Epidemiological surveillance (2009-2012) was performed in Campinas, State of São Paulo, Brazil. Results In 2009, Leishmania chagasi was positively identified in four dogs. Entomological research and three serological studies (2010-2012) were undertaken as monitoring measures; these approaches revealed a moderate prevalence of Leishmania present in 4% of the canine population. Nyssomyia whitmani and Lutzomyia longipalpis were the predominant species identified. Conclusions Detection of an AVL outbreak in dogs in an area with an evolving natural landscape containing sand flies is crucial for control programs.

Deltamethrin pyrethroid susceptibility characterization of Triatoma sordida Stål, 1859 (Hemiptera: Reduviidae) populations in the Northern Region of Minas Gerais, Brazil

Introduction Triatoma sordida is the most captured Triatomine species in the Brazilian artificial environment. In 2008, the discovery of three Triatomine populations with altered susceptibilities to deltamethrin highlighted the importance of investigating the genetic potential for resistance in triatomines. The purpose of this study was to characterize the susceptibility to deltamethrin of peridomestic T. sordida populations in Minas Gerais, Brazil. Methods A susceptibility reference lineage derived from Uberaba, Minas Gerais, Brazil was used. Serial dilutions of deltamethrin were prepared and applied to the dorsal abdomen of first instar nymphs. The control group received only pure acetone. Mortality was evaluated after 72h. Qualitative tests assessed mortality in response to a diagnostic dose of 1xLD99 of the susceptibility reference lineage. Results Susceptibility profile characterization of T. sordida populations revealed resistance ratios (RR50s) ranging from 0.42 to 3.94. The percentage mortality in response to the diagnostic dose varied from 70% to 100%. A comparison of the results obtained in the quantitative and qualitative assays demonstrated a lack of correspondence for some populations. Conclusions We demonstrated that only T. sordida populations that present a RR50>1.0 have altered susceptibility, and the execution of simultaneous field and laboratory tests is required to understand the actual effect of vector control. A possible cause of the observed resistance ratios might be the continuous use of pyrethroids in Brazil since the 1980s.

Colistin and anti-Gram-positive bacterial agents against Acinetobacter baumannii

Introduction Acinetobacter baumannii has attained an alarming level of resistance to antibacterial drugs. Clinicians are now considering the use of older agents or unorthodox combinations of licensed drugs against multidrug-resistant strains to bridge the current treatment gap. We investigated the in vitro activities of combination treatments that included colistin with vancomycin, norvancomycin or linezolid against multidrug-resistant Acinetobacter baumannii. Methods The fractional inhibitory concentration index and time-kill assays were used to explore the combined effects of colistin with vancomycin, norvancomycin or linezolid against 40 clinical isolates of multidrug-resistant Acinetobacter baumannii. Transmission electron microscopy was performed to evaluate the interactions in response to the combination of colistin and vancomycin. Results The minimum inhibitory concentrations (MICs) of vancomycin and norvancomycin for half of the isolates decreased below the susceptibility break point, and the MIC of linezolid for one isolate was decreased to the blood and epithelial lining fluid concentration using the current dosing regimen. When vancomycin or norvancomycin was combined with subinhibitory doses of colistin, the multidrug-resistant Acinetobacter baumannii test samples were eradicated. Transmission electron microscopy revealed that subinhibitory doses of colistin were able to disrupt the outer membrane, facilitating a disruption of the cell wall and leading to cell lysis. Conclusions Subinhibitory doses of colistin significantly enhanced the antibacterial activity of vancomycin, norvancomycin, and linezolid against multidrug-resistant Acinetobacter baumannii.

Epidemiological and environmental aspects of visceral leishmaniasis in children under 15 years of age between 2007 and 2012 in the City of Araguaína, State of Tocantins, Brazil

Introduction Visceral leishmaniasis (VL) stands out as a zoonosis observed on four continents and also in urban expansion zones in several regions of Brazil. Methods A cross-sectional epidemiological study of VL cases in children under 15 years of age in the period from 2007 to 2012. Clinical data were gathered from medical reports; meteorological data were obtained at the Meteorological Measurement Department of UFT. Environmental variables were divided into two periods, rainy and dry. Results The study revealed no difference by gender (p=0.67) among the 821 patients. However, the most affected age group was between one and five years of age (58.6%; p<0.01); the highest prevalence of the disease (99.03%; p<0.01) occurred in urban zones; and the most affected ethnic group (85.5%; p<0.01) was mixed race. The highest incidence coefficients in this population occurred in 2007 and 2008 (578.39/100,000 inhabitants; 18.5/100,000 inhabitants, respectively), whereas the highest lethality coefficients occurred in 2008 and 2011 (0.85/100 deaths). There was no significant correlation between average rainfall and the number of VL cases. The correlation between temperature and number of VL cases was negative (r = -0.4039; p<0.01). Conclusions In Araguaína, visceral leishmaniasis in children under 15 years is an urban-based endemic disease distributed across all districts of the city wherein temperature as an environmental factor, a higher prevalence in mixed race children between one and five years of age, and a high incidence coefficient all strongly contribute to child mortality.