PCR-RFLP of 16S ribosomal DNA to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples

INTRODUCTION: This study aimed to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples by PCR-RFLP. METHODS: Fifty-two strains identified by conventional biochemical exams were submitted to PCR amplification and digested with HinfI. Only 20 (38.5%) of the 52 strains showed a DNA pattern expected for E. gallinarum and E. casseliflavus. RESULTS: Analysis of the results of this study showed that E. gallinarum and E. casseliflavus are occasionally erroneously identified and confirmed the potential application of 16S rDNA analysis for accurate identification of these species. CONCLUSIONS: A correct identification is important to distinguish between intrinsic and acquired vancomycin resistance.

PCR detection of multiple human herpesvirus DNA in saliva from HIV-infected individuals in Teresina, State of Piauí, Brazil

INTRODUCTION: Human herpesviruses are frequently associated with orofacial diseases in humans (HSV-1, EBV, CMV and HHV-8), some can also cause systemic disease (CMV and HHV-8). The transmission of these viruses occurs by contact with infected secretions, especially saliva. Human immunodeficiency virus infection is associated with an increased risk of HHVs and related diseases. METHODS: This work aimed to detect HSV-1, EBV, CMV and HHV-8 DNA in saliva of HIV-infected patients from Teresina, northeast Brazil, by PCR and compare these findings with age and sex matched HIV-seronegative individuals. RESULTS: No difference in prevalence was verified between HHV detection in the saliva of HIV-seropositive individuals and controls. The individual frequencies of these viruses in these two populations were different. HIV seropositivity correlated positively with the presence of CMV (OR: 18.2, p= 0.00032) and EBV (OR: 3.44, p= 0.0081). No association between CD4 counts and the prevalence of HHVs in the saliva was observed; however, a strong association was determined between seropositivity and the presence of multiple HHV DNAs in saliva (OR: 4.83, p = 0.0028). CONCLUSIONS: These findings suggest the asymptomatic salivary shedding of HHVs is a common event between HIV-seropositive and seronegative individuals from Teresina, Piauí, Brazil, and, especially for HIV-seropositive patients, saliva is a risk factor for the acquisition/transmission of multiple HHVs.

Comparison among three polymerase chain reaction assays on detection of DNA from Leishmania in biological samples from patients with american cutaneous leishmaniasis

INTRODUCTION: The study analyzed positivity of polymerase chain reaction (PCR) on detection of DNA from Leishmania in patients' samples. METHODS: Extracted DNA was submitted to L150/L152, 13Y/13Z, and seminested PCR (snPCR). RESULTS: Results were evidenced by bands of approximately 120, 720, and 670 bp for L150/L152, 13Y/13Z, and snPCR, respectively. L150/L152, 13Y/13Z, and snPCR positivity indexes were 76.9, 56.4, and 9.2 (p>0.05), respectively, for suspected and 93.7, 68.7, and 84.4 (p<0.05), respectively, for confirmed. CONCLUSIONS: Preliminary results showed that these assays, mainly L150/L152 and snPCR, can detect Leishmania DNA and carry potential on laboratory diagnosis of leishmaniasis.

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.

A comparison of four DNA extraction protocols for the analysis of urine from patients with visceral leishmaniasis

Introduction Polymerase chain reaction (PCR) may offer an alternative diagnostic option when clinical signs and symptoms suggest visceral leishmaniasis (VL) but microscopic scanning and serological tests provide negative results. PCR using urine is sensitive enough to diagnose human visceral leishmaniasis (VL). However, DNA quality is a crucial factor for successful amplification. Methods A comparative performance evaluation of DNA extraction methods from the urine of patients with VL using two commercially available extraction kits and two phenol-chloroform protocols was conducted to determine which method produces the highest quality DNA suitable for PCR amplification, as well as the most sensitive, fast and inexpensive method. All commercially available kits were able to shorten the duration of DNA extraction. Results With regard to detection limits, both phenol: chloroform extraction and the QIAamp DNA Mini Kit provided good results (0.1 pg of DNA) for the extraction of DNA from a parasite smaller than Leishmania (Leishmania) infantum (< 100fg of DNA). However, among 11 urine samples from subjects with VL, better performance was achieved with the phenol:chloroform method (8/11) relative to the QIAamp DNA Mini Kit (4/11), with a greater number of positive samples detected at a lower cost using PCR. Conclusion Our results demonstrate that phenol:chloroform with an ethanol precipitation prior to extraction is the most efficient method in terms of yield and cost, using urine as a non-invasive source of DNA and providing an alternative diagnostic method at a low cost.

Occult HBV infection status among chronic hepatitis C and hemodialysis patients in Northeastern Egypt: regional and national overview

INTRODUCTION: Occult hepatitis B infection (OBI) is considered to be one of the major risks for patients suffering from end-stage renal disease (ESRD) on regular hemodialysis (HD) and patients with chronic hepatitis C virus (HCV) infection. This study compared the prevalence of OBI among these two high-risk groups in the Suez Canal region, Northeastern Egypt, to obtain a better national overview of the magnitude of OBI in this region. METHODS: Serum samples were collected from 165 HD patients and 210 chronic HCV-infected patients. Anti-HCV antibody, hepatitis B surface antigen (HBsAg), total hepatitis B core (anti-HBc) antibody, and hepatitis B surface antibody (anti-HBs) were detected by enzyme-linked immunosorbent assay (ELISA). HCV RNA was detected using a quantitative real-time RT-PCR assay, and HBV was detected using a nested PCR. RESULTS: All patients were negative for HBsAg. A total of 49.1% and 25.2% of the patients in the HD and HCV groups, respectively, were anti-HBc-positive. In addition, more anti-HBs-positive patients were detected in the HD group compared to the HCV group (52.1% and 11.4%, respectively). Three cases were positive for HBV DNA in the HD group, while eighteen positive cases were detected in the HCV group. Both study groups showed significant differences in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) level as well as anti-HBc, anti-HBs and HBV-DNA positivity. CONCLUSIONS: OBI was more prevalent among chronic HCV patients than HD patients in the Suez Canal region, Egypt, with rates of 8.5% and 1.8%, respectively. However, more precise assessment of this infection requires regular patient follow-up using HBV DNA detection methods.

Detection of Leishmania (Viannia) DNA in leucocytes from the blood of patients with cutaneous leishmaniasis

ABSTRACTINTRODUCTION:Cutaneous leishmaniasis (CL) is a serious and global public health issue, with the potential of developing a mucosal form, occurring as subclinical cases, and showing recurrence despite previous treatment.METHODS:Polymorphonuclear and mononuclear DNA obtained from 49 patients was subjected to polymerase chain reaction for detection of Leishmania (Viannia).RESULTS:DNA was detected in mononuclear cells from two patients with active primary lesions positive for CL, with infection periods of 3 and 6 months, respectively.CONCLUSIONS:The DNA of Leishmania (Viannia) indicates probable parasite dissemination possibly explaining subclinical case emergence, lesion recurrence, and mucosal lesion appearance.

Single-tube nested PCR assay with in-house DNA extraction for Mycobacterium tuberculosis detection in blood and urine

Abstract: INTRODUCTION : Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. METHODS: Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). RESULTS: STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary). CONCLUSIONS: STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.

Extraction of Trypanosoma cruzi DNA from food: a contribution to the elucidation of acute Chagas disease outbreaks

Abstract: INTRODUCTION: Before 2004, the occurrence of acute Chagas disease (ACD) by oral transmission associated with food was scarcely known or investigated. Originally sporadic and circumstantial, ACD occurrences have now become frequent in the Amazon region, with recently related outbreaks spreading to several Brazilian states. These cases are associated with the consumption of açai juice by waste reservoir animals or insect vectors infected with Trypanosoma cruzi in endemic areas. Although guidelines for processing the fruit to minimize contamination through microorganisms and parasites exist, açai-based products must be assessed for quality, for which the demand for appropriate methodologies must be met. METHODS: Dilutions ranging from 5 to 1,000 T. cruzi CL Brener cells were mixed with 2mL of acai juice. Four Extraction of T. cruzi DNA methods were used on the fruit, and the cetyltrimethyl ammonium bromide (CTAB) method was selected according to JRC, 2005. RESULTS: DNA extraction by the CTAB method yielded satisfactory results with regard to purity and concentration for use in PCR. Overall, the methods employed proved that not only extraction efficiency but also high sensitivity in amplification was important. CONCLUSIONS: The method for T. cruzi detection in food is a powerful tool in the epidemiological investigation of outbreaks as it turns epidemiological evidence into supporting data that serve to confirm T. cruzi infection in the foods. It also facilitates food quality control and assessment of good manufacturing practices involving acai-based products.

Human identification and analysis of DNA in bones

The introduction of molecular biology techniques, especially of DNA analysis, for human identification is a recent advance in legal medicine. Substantial effort has continuously been made in an attempt to identify cadavers and human remains after wars, socio-political problems and mass disasters. In addition, because of the social dynamics of large cities, there are always cases of missing people, as well as unidentified cadavers and human remains that are found. In the last few years, there has also been an increase in requests for exhumation of human remains in order to determine genetic relationships in civil suits and court action. The authors provide an extensive review of the literature regarding the use of this new methodology for human identification of ancient or recent bones.

Chlamydia pneumoniae and atherosclerosis. Identification of bacterial DNA in the arterial wall

OBJECTIVE: The intracellular Gram-negative bacterium Chlamydia pneumoniae has been associated with atherosclerosis. The presence of Chlamydia pneumoniae has been investigated in fragments of the arterial wall with a technique for DNA identification. METHODS: Arterial fragments obtained from vascular surgical procedures in 58 patients were analyzed. From these patients, 39 were males and the mean age was 65±6 years. The polymerase chain reaction was used to identify the bacterial DNA with a pair of primers that codify the major outer membrane protein (MOMP) of Chlamydia pneumoniae. The amplified product was visualized by electrophoresis in the 2% agarose gel stained with ethidium bromide, and it was considered positive when migrating in the band of molecular weight of the positive controls. RESULTS: Seven (12%) out of the 58 patients showed positive results for Chlamydia pneumoniae. CONCLUSION: DNA from Chlamydia pneumoniae was identified in the arterial wall of a substantial number of patients with atherosclerosis. This association, which has already been described in other countries, corroborates the evidence favoring a role played by Chlamydia pneumoniae in atherogenesis.

End-systolic pressure-diameter relation of the left ventricle during transient and sustained elevations of blood pressure

OBJECTIVE: To assess the effect of transient and sustained variations in cardiac load on the values of the end-systolic pressure-diameter relation (ESPDR) of the left ventricle. METHODS: We studied 13 dogs under general anesthesia and autonomic blockade. Variations of cardiac loads were done by elevation of blood pressure by mechanical constriction of the aorta. Two protocols were used in each animal: gradual peaking and decreasing pressure variation, the "transient arterial hypertension protocol" (TAH), and a quick and 10 min sustained elevation, the "sustained arterial hypertension protocol"(SAH). Then, we compared the ESDR in these two situations. RESULTS: Acute elevation of arterial pressure, being it "transitory" or "sustained", did not alter the heart frequency and increased similarly the preload and after load. However, they acted differently in end systolic pressure-diameter relation. It was greater in the SAH than TAH protocol, 21.0±7.3mmHg/mm vs. 9.2±1.2mmHg/mm (p<0.05). CONCLUSION: The left ventricular ESPDR values determined during sustained pressure elevations were higher than those found during transient pressure elevations. The time-dependent activation of myocardial contractility associated with the Frank-Starling mechanism is the major factor in inotropic stimulation during sustained elevations of blood pressure, determining an increase in the ESPDR values.

Evaluation of Electrocardiographic T-peak to T-end Interval in Subjects with Increased Epicardial Fat Tissue Thickness

Abstract Background: The association between periatrial adiposity and atrial arrhythmias has been shown in previous studies. However, there are not enough available data on the association between epicardial fat tissue (EFT) thickness and parameters of ventricular repolarization. Thus, we aimed to evaluate the association of EFT thickness with indices of ventricular repolarization by using T-peak to T-end (Tp-e) interval and Tp-e/QT ratio. Methods: The present study included 50 patients whose EFT thickness ≥ 9 mm (group 1) and 40 control subjects with EFT thickness < 9 mm (group 2). Transthoracic echocardiographic examination was performed in all participants. QT parameters, Tp-e intervals and Tp-e/QT ratio were measured from the 12-lead electrocardiogram. Results: QTd (41.1 ± 2.5 vs 38.6 ± 3.2, p < 0.001) and corrected QTd (46.7 ± 4.7 vs 43.7 ± 4, p = 0.002) were significantly higher in group 1 when compared to group 2. The Tp-e interval (76.5 ± 6.3, 70.3 ± 6.8, p < 0.001), cTp-e interval (83.1 ± 4.3 vs. 76±4.9, p < 0.001), Tp-e/QT (0.20 ± 0.02 vs. 0.2 ± 0.02, p < 0.001) and Tp-e/QTc ratios (0.2 ± 0.01 vs. 0.18 ± 0.01, p < 0.001) were increased in group 1 in comparison to group 2. Significant positive correlations were found between EFT thickness and Tp-e interval (r = 0.548, p < 0.001), cTp-e interval (r = 0.259, p = 0.01), and Tp-e/QT (r = 0.662, p < 0.001) and Tp-e/QTc ratios (r = 0.560, p < 0.001). Conclusion: The present study shows that Tp-e and cTp-e interval, Tp-e/QT and Tp-e/QTc ratios were increased in subjects with increased EFT, which may suggest an increased risk of ventricular arrhythmia.