EVALUATION OF FOUR DIFFERENT DNA EXTRACTION METHODS IN COAGULASE-NEGATIVE STAPHYLOCOCCI CLINICAL ISOLATES

Currently there are several methods to extract bacterial DNA based on different principles. However, the amount and the quality of the DNA obtained by each one of those methods is highly variable and microorganism dependent, as illustrated by coagulase-negative staphylococci (CoNS) which have a thick cell wall that is difficult to lyse. This study was designed to compare the quality and the amount of CoNS DNA, extracted by four different techniques: two in-house protocols and two commercial kits. DNA amount and quality determination was performed through spectrophotometry. The extracted DNA was also analyzed using agarose gel electrophoresis and by PCR. 267 isolates of CoNS were used in this study. The column method and thermal lyses showed better results with regard to DNA quality (mean ratio of A260/280 = 1.95) and average concentration of DNA (), respectively. All four methods tested provided appropriate DNA for PCR amplification, but with different yields. DNA quality is important since it allows the application of a large number of molecular biology techniques, and also it's storage for a longer period of time. In this sense the extraction method based on an extraction column presented the best results for CoNS.

FALSE-NEGATIVE DENGUE CASES IN RORAIMA, BRAZIL: AN APPROACH REGARDING THE HIGH NUMBER OF NEGATIVE RESULTS BY NS1 AG KITS

Serum samples from 150 NS1-negative (Platelia ELISA) patients presumptively diagnosed with dengue were analyzed by the TaqMan probed real-time reverse transcription PCR (TaqMan qRT-PCR) method. The qRT-PCR positive samples were tested for serotype by semi-nested RT-PCR and a qualitative immunochromatographic assay for IgG and IgM. Molecular detection methods showed 33 (22%) positive samples out of 150 NS1-antigen negative samples. Of these, 72% were collected up to day 2 after the onset of symptoms, when diagnostic sensitivity of NS1-antigen test assays is significantly enhanced. Most of the cases were not characterized as secondary infection. Twenty-eight samples were successfully serotyped, 75% of which for DENV-4, 14% for DENV-2, 7% for DENV-3 and 4% for DENV-1. These findings reaffirm the hyperendemic situation of the state of Roraima and suggest a lower sensitivity of the NS1 test, mainly when DENV-4 is the predominant serotype. Health care providers should therefore be aware of samples tested negative by NS1 antigen assays, especially when clinical symptoms and other laboratory data results show evidence of dengue infection.

DISSEMINATED FUNGAL INFECTION WITH ADRENAL INVOLVEMENT: REPORT OF TWO HIV NEGATIVE BRAZILIAN PATIENTS

Paracoccidioidomycosis and histoplasmosis are systemic fungal infections endemic in Brazil. Disseminated clinical forms are uncommon in immunocompetent individuals. We describe two HIV-negative patients with disseminated fungal infections, paracoccidioidomycosis and histoplasmosis, who were diagnosed by biopsies of suprarenal lesions. Both were treated for a prolonged period with oral antifungal agents, and both showed favorable outcomes.

INVESTIGATION OF BIOFILM FORMATION IN COAGULASE-NEGATIVE STAPHYLOCOCCI ISOLATED FROM PLATELET CONCENTRATE BAGS

Platelet Concentrates (PCs) are the blood components with the highest rate of bacterial contamination, and coagulase-negative staphylococci (CoNS) are the most frequently isolated contaminants. This study investigated the biofilm formation of 16 contaminated units out of 691 PCs tested by phenotypic and genotypic methods. Adhesion in Borosilicate Tube (ABT) and Congo Red Agar (CRA) tests were used to assess the presence of biofilm. The presence of icaADC genes was assessed by means of the Polymerase Chain Reaction (PCR) technique. With Vitek(r)2, Staphylococcus haemolyticus was considered the most prevalent CoNS (31.25%). The CRA characterized 43.8% as probable biofilm producers, and for the ABT test, 37.5%. The icaADC genes were identified in seven samples by the PCR. The ABT technique showed 85.7% sensitivity and 100% specificity when compared to the reference method (PCR), and presented strong agreement (k = 0.8). This study shows that species identified as PCs contaminants are considered inhabitants of the normal skin flora and they might become important pathogens. The results also lead to the recommendation of ABT use in laboratory routine for detecting biofilm in CoNS contaminants of PCs.

Comparative clinical and ultrasound study of egg-negative and egg-positive individuals from Schistosoma mansoni low morbidity endemic areas, and hospitalized patients with hepatosplenic disease

Two hundred and twenty three subjects from a Schistosoma mansoni low morbidity endemic area and nine hospitalized hepatosplenic patients were submitted to stool test and clinical examination and abdomen ultrasound assessments. According to stool examination and ultrasound results, they were grouped as follows: G1 - 63 Schistosoma mansoni egg-negative individuals; G2 - 141 egg-positive patients and without evidence of periportal fibrosis; G3 - 19 egg-positive patients with periportal echogenicity (3-6mm); and G4 - 9 hepatosplenic patients with periportal echogenicity (> 6mm). Hepatomegaly detected by physical examination of the abdomen evaluated in the midclavicular line was verified in G1, G2 and G3, respectively, in 11.1, 12.1 and 26.3%. In G1, G2 and G3, periportal thickening occurred only in schistosomal patients (8.5%). Mild pathological alterations in patients that cannot yet be detected by clinical examination were detectable in the liver by ultrasound and can be due to fibrosis. The degree of mild periportal fibrosis was diminished in 57.9% of patients 12 months after treatment of schistosomiasis with oxamniquine. At ultrasonography, the mean liver left lobe measurement of G3 was larger than that of G1, and that of G4 larger than that of G1 and G2. The mean size of the spleen of G4 was significantly larger than that of the other three groups, and that of G3 larger than that of G1 and G2.

Characterization of a new coagulase-negative Staphylococcus species (Staphylococcus pettenkoferi) isolated from blood cultures from a hospitalized patient in Porto Alegre, Brazil

INTRODUCTION: Staphylococcus pettenkoferi was originally isolated and described by Trülzsch et al (2002). In this study, we characterized two isolates of this newly described species. METHODS: Blood cultures were initially processed using the BacT/ALERT® device, and the isolates were initially characterized using the Vitek2 identification system. RESULTS: The initial characterization revealed slow-growing Gram-positive cocci that formed opaque colonies on sheep blood agar. Other phenotypic/genotypic tests were performed. CONCLUSIONS: We would like to emphasize that this new staphylococcus species is phenotypically similar to other CoNS, especially S. auricularis. This could potentially lead to misidentification of these uncommon species.

Cryptococcus gattii meningoencephalitis in an HIV-negative patient from the Peruvian Andes

We report a case of an immunocompetent Peruvian patient from the Andes with a one-month history of meningoencephalitis. Cryptococcus gattii was identified from a cerebrospinal fluid culture through assimilation of D-proline and D-tryptophan as the single nitrogen source. Initially, the patient received intravenous antifungal therapy with amphotericin B. The patient was discharged 29 days after hospitalization and continued with oral fluconazole treatment for ten weeks. During this period, the patient showed clinical improvement with slight right-side residual weakness. Through this case report, we confirm the existence of this microorganism as an infectious agent in Peru.

Clinical, demographic and epidemiological characteristics of patients with hepatitis B followed at a university hospital in southeastern Brazil: predominance of HBeAg negative cases

INTRODUCTION: Hepatitis B is common in Brazil, although there are regional differences regarding the degree of endemicity, the most frequent forms of transmission and the presence of different evolutive stages of chronic disease. The present study aimed to determine the clinical, demographic and epidemiological characteristics of patients chronically infected with hepatitis B virus (HBV) residing in the Ribeirão Preto region, southeastern Brazil. METHODS: A total of 529 medical records of individuals with HBV monoinfection were reviewed. RESULTS: More than 60% of the subjects were males, with a mean age of 38 years-old. The HBeAg-negative serological pattern was verified in 84.4% of the patients, among whom the risk of vertical/intrafamily transmission was 43.2% (p = 0.02). The consumption of alcohol in amounts exceeding 20g a day was observed in 21.3% of the subjects and was more frequent among men (33%) (p < 0.001). Among patients with cirrhosis, 54.1% were alcohol abusers (p = 0.04), all of them males. The presence of cirrhosis was more frequent in the HBeAg-positive group (24.4%) than in the HBeAg-negative group (10.2%) (p < 0.001). CONCLUSIONS: High proportions of HBV-infected subjects with an HBeAg-negative pattern were observed, with a higher risk of vertical/intrafamily transmission. Alcohol abuse was associated with male subjects and with cirrhosis of the liver in this group. A tendency toward an increase in the number of HBeAg-negative cases was observed over time.

Prevalence of enterotoxin-encoding genes and antimicrobial resistance in coagulase-negative and coagulase-positive Staphylococcus isolates from black pudding

INTRODUCTION: Staphylococcal species are pathogens that are responsible for outbreaks of foodborne diseases. The aim of this study was to investigate the prevalence of enterotoxin-genes and the antimicrobial resistance profile in staphylococcus coagulase-negative (CoNS) and coagulasepositive (CoPS) isolates from black pudding in southern Brazil. METHODS: Two hundred typical and atypical colonies from Baird-Parker agar were inoculated on mannitol salt agar. Eighty-two mannitol-positive staphylococci were submitted to conventional biochemical tests and antimicrobial susceptibility profiling. The presence of coagulase (coa) and enterotoxin (se) genes was investigated by polymerase chain reaction. RESULTS: The isolates were divided into 2 groups: 75.6% (62/82) were CoNS and 24.4% (20/82) were CoPS. The biochemical tests identified 9 species, of which Staphylococcus saprophyticus (37.8%) and Staphylococcus carnosus (15.9%) were the most prevalent. Antimicrobial susceptibility tests showed resistance phenotypes to antibiotics widely administered in humans, such as gentamicin, tetracycline, chloramphenicol, and erythromycin. The coa gene was detected in 19.5% (16/82) of the strains and 4 polymorphic DNA fragments were observed. Five CoNS isolates carrying the coa gene were submitted for 16S rRNA sequencing and 3 showed similarity with CoNS. Forty strains were positive for at least 1 enterotoxin-encoding gene, the genes most frequently detected were sea (28.6%) and seb (27.5%). CONCLUSIONS: The presence of antimicrobial resistant and enterotoxin-encoding genes in staphylococci isolates from black pudding indicated that this fermented food may represent a potential health risk, since staphylococci present in food could cause foodborne diseases or be a possible route for the transfer of antimicrobial resistance to humans.

Hepatitis B revaccination for healthcare workers who are anti-HBs-negative after receiving a primary vaccination series

INTRODUCTION: This study aimed to evaluate the response to hepatitis B (HB) revaccination of healthcare workers (HCW) who are negative for antibodies to HB surface antigen (anti-HBs) after a complete vaccination series. METHODS: HCW whose anti-HBs test was performed > 90 days after a HB vaccination course were given a 4th dose. A post-vaccination test was done within 30 to 90 days. RESULTS: One hundred and seventy HCW were enrolled: 126 (74.1%) were anti-HBs-positive after the 4th dose. CONCLUSIONS: Rechecking anti-HBs after the 4th HB vaccine dose is a practical approach in case of post-vaccination tests performed >90 days after the full vaccination course.

Prevalence of resistance phenotypes in Staphylococcus aureus and coagulase-negative isolates of venous ulcers of primary healthcare patients

INTRODUCTION: In venous ulcers, the presence of Staphylococcus aureus and coagulase-negative staphylococcus resistance phenotypes can aggravate and limit the choices for treatment. METHODS: Staphylococcus isolated from 69 patients (98 ulcers) between October of 2009 and October of 2010 were tested. The macrolide, lincosamide, streptogramin B (MLS B) group resistance phenotype detection was performed using the D-test. Isolates resistant to cefoxitin and/or oxacillin (disk-diffusion) were subjected to the confirmatory test to detect minimum inhibitory concentration (MIC), using oxacillin strips (E-test®). RESULTS: The prevalence of S. aureus was 83%, and 15% of coagulase-negative staphylococcus (CoNS). In addition were detected 28% of methicillin-resistant Staphylococcus aureus (MRSA) and 47% of methicillin-resistant coagulase-negative staphylococcus (MRCoNS). Among the S. aureus, 69.6% were resistant to erythromycin, 69.6% to clindamycin, 69.6% to gentamicin, and 100% to ciprofloxacin. Considering the MRSA, 74% were highly resistant to oxacillin, MIC ≥ 256µg/mL, and the MLS Bc constitutive resistance predominated in 65.2%. Among the 20 isolates sensitive to clindamycin, 12 presented an inducible MLS B phenotype. Of the MRCoNS, 71.4%were resistant to erythromycin, ciprofloxacin and gentamicin. Considering the isolates positive for β-lactamases, the MIC breakpoint was between 0.5 and 2µg/mL. CONCLUSIONS: The results point to a high occurrence of multi-drug resistant bacteria in venous ulcers in primary healthcare patients, thus evidencing the need for preventive measures to avoid outbreaks caused by multi-drug resistant pathogens, and the importance of healthcare professionals being able to identifying colonized versus infected venous ulcers as an essential criteria to implementing systemic antibacterial therapy.

Puerperal brain cryptococcoma in an HIV-negative woman successfully treated with fluconazole: a case report

Cryptococcus spp. cerebral abscesses are uncommon in immunocompetent subjects. The recommended induction treatment is the administration of amphotericin B plus flucytosine combined with resection for lesions ≥3cm. In this paper, we describe an HIV-negative woman diagnosed with a large cryptococcoma in the immediate postpartum period. The lesion was not resected, and due to amphotericin B intolerance, she received an extended course of fluconazole monotherapy. There was no disease recurrence during the 4 years of follow-up. The abrupt onset of her symptoms following delivery suggests that she developed a postpartum immune reconstitution syndrome. This case also demonstrates that in specific situations fluconazole monotherapy can be attempted in immunocompetent patients with cryptococcoma.

Pharmacokinetic/pharmacodynamic target attainment of intravenous β-lactam regimens against Gram-negative bacteria isolated in a Brazilian teaching hospital

ABSTRACTINTRODUCTION: Monte Carlo simulations have been used for selecting optimal antibiotic regimens for treatment of bacterial infections. The aim of this study was to assess the pharmacokinetic and pharmacodynamic target attainment of intravenous β-lactam regimens commonly used to treat bloodstream infections (BSIs) caused by Gram-negative rod-shaped organisms in a Brazilian teaching hospital.METHODS: In total, 5,000 patients were included in the Monte Carlo simulations of distinct antimicrobial regimens to estimate the likelihood of achieving free drug concentrations above the minimum inhibitory concentration (MIC; fT > MIC) for the requisite periods to clear distinct target organisms. Microbiological data were obtained from blood culture isolates harvested in our hospital from 2008 to 2010.RESULTS: In total, 614 bacterial isolates, including Escherichia coli, Enterobacterspp., Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa, were analyzed Piperacillin/tazobactam failed to achieve a cumulative fraction of response (CFR) > 90% for any of the isolates. While standard dosing (short infusion) of β-lactams achieved target attainment for BSIs caused by E. coliand Enterobacterspp., pharmacodynamic target attainment against K. pneumoniaeisolates was only achieved with ceftazidime and meropenem (prolonged infusion). Lastly, only prolonged infusion of high-dose meropenem approached an ideal CFR against P. aeruginosa; however, no antimicrobial regimen achieved an ideal CFR against A. baumannii.CONCLUSIONS:These data reinforce the use of prolonged infusions of high-dose β-lactam antimicrobials as a reasonable strategy for the treatment of BSIs caused by multidrug resistant Gram-negative bacteria in Brazil.