Alice Beatriz Mombach Pinheiro Machado

Group B Streptococcus detection: comparison of PCR assay and culture as a screening method for pregnant women

Streptococcus agalactiae or group B Streptococcus (GBS) is one of the most important causal agents of serious neonatal infections. Numerous assays have been evaluated for GBS screening in order to validate a fast and efficient method. The aim of this study was to compare the culture technique (established as the gold standard) with the molecular method of polymerase chain reaction (PCR) with specific primers (atr gene). Two hundred and sixty-three samples were analyzed. Vaginal samples were collected, according to the Centers for Disease Control and Prevention (CDC) recommendations, from women over 35 weeks of pregnancy at Hospital de Clínicas de Porto Alegre (HCPA). Two different extraction methods were tested in all samples collected. PCR technique yielded 71 (26.99%) positive results. Sensitivity and specificity for PCR were 100% and 86.88%, respectively. PCR demonstrated a shorter turnaround time than the culture. The molecular methodology proved to be a useful screening for GBS, allowing effective treatment to be initiated in shorter time to prevent newborn infection.

Distribution of erm genes and low prevalence of inducible resistance to clindamycin among staphylococci isolates

INTRODUCTION Resistance to macrolides, lincosamides and streptogramins B (MLS(B) antibiotics) in staphylococci may be due to modification in ribosomal target methylase encoded by erm genes. The expression of MLS(B) resistance lead to three phenotypes, namely constitutive resistance (cMLS(B)), inducible resistance (iMLS(B)), and resistance only to macrolides and streptogramins B (MS(B)). The iMLS(B) resistance is the most difficult to detect in the clinical laboratory. OBJECTIVE This study investigated the expression of MLS(B) resistance and the prevalence of the erm genes among 152 clinical isolates of Staphylococcus aureus and coagulase-negative Staphylococcus (CNS) from Hospital de Clínicas de Porto Alegre. METHODS Primary MLS(B) resistance was detected by the disk diffusion method. Isolates with iMLS(B) phenotype were tested by double-disk induction method. All isolates were tested by a genotypic assay, PCR with specific primers. RESULTS A total of 46.7% of staphylococci were positive for cMLS(B); 3.3% for iMLS(B) and 3.3% for MS(B). One or more erm genes were present in 50.1% of isolates. The gene ermA was detected in 49 isolates, ermC in 29 and ermB in 3. CONCLUSION The prevalence of the ermA, ermB and ermC genes were 29.6%, 17.1% and 0.66% respectively, and constitutive resistance was the most frequent as compared to the other two phenotypes.

Vancomycin MIC for Methicillin-Resistant Coagulase-Negative Staphylococcus Isolates: Evaluation of the Broth Microdilution and Etest Methods

ABSTRACT Vancomycin MIC results were determined by the broth microdilution (BMD) method and by Etest using 130 methicillin-resistant coagulase-negative staphylococcus bloodstream isolates obtained from a tertiary hospital. The majority (98.5%) of MIC results determined by BMD were ≤1 μg/ml, in contrast to MIC results determined by Etest (72.3% were ≥1.5 μg/ml). The MICs obtained by Etest were, in general, 1- to 2-fold higher than the MICs obtained by BMD.

Optimization of one-step duplex real-time RT-PCR for detection of influenza and respiratory syncytial virus in nasopharyngeal aspirates.

Viruses are major contributors to acute respiratory infection-related morbidity and mortality worldwide. The influenza (IF) viruses and human respiratory syncytial virus (RSV) play a particularly important role in the etiology of acute respiratory infections. This study sought to standardize a one-step duplex real-time RT-PCR technique to optimize diagnosis of IFA/IFB and RSVA/RSVB infection. Viral RNA was extracted with the commercially available QIAamp Mini Kit according to manufacturer instructions. RT-PCR was performed with primers to the matrix protein gene of IFA, the hemagglutinin gene of IFB and the N gene of RSVA and RSVB. The limits of detection were 1 copy/μL for IFA, 10 copies/μL for IFB, 5 copies/μL for RSVA, and 250 copies/μL for RSVB. The specificity of RT-PCR was determined by comparison against a panel of several respiratory pathogens. RT-PCR and indirect immunofluorescence (IIF) were compared in a sample of 250 nasopharyngeal aspirates (NPAs) collected during the year 2010. RT-PCR was more sensitive than IIF and able to detect viral co-infections. In summary, RT-PCR optimized for IFA/IFB and RSVA/RSVB is sensitive and specific for these viral agents and is therefore useful for assessment of the etiology of respiratory infections, whether for clinical or epidemiological purposes.

High prevalence of methicillin-resistant Staphylococcus aureus with SCCmec type III in cystic fibrosis patients in southern, Brazil

INTRODUCTION Bacterial colonization of the lungs is the main cause of morbidity in cystic fibrosis (CF). Pathogens such as Staphylococcus aureus are very well adapted to the pulmonary environment and may persist for years in the same patient. Genetic determinants of these bacteria, such as the presence of SCCmec have recently emerged as a problem in this population of patients. METHODS Staphylococcus aureus isolates obtained from different clinical materials coming from CF and non-CF patients attended at a cystic fibrosis reference hospital were compared according to SCCmec type and antibiotic susceptibility profile. RESULTS Three hundred and sixty-four single-patient Staphylococcus aureus isolates were collected, of which 164 (45%) were from CF patients. Among the latter, 57/164 (44.5%) were MRSA, and among the non-CF patients, 89/200 (35%) were MRSA. Associated pathogens were found in 38 CF patients. All 57 MRSA from CF patients harbored the multiresistant cassette type III. In contrast, 31/89 MRSA from non-CF patients harbored SCCmec type I (35%) and 44/89 harbored type III (49%). The antibiotic susceptibility pattern was similar between CF and non-CF patients. CONCLUSIONS The high prevalence of multiresistant SCCmec type III among CF patients compared with non-CF patients in our institution may make it difficult to control disease progression through antibiotic therapy for promoting the survival of this kind of patient.

Genetic similarity of Burkholderia cenocepacia from cystic fibrosis patients

Burkholderia cenocepacia may cause serious infections in patients with cystic fibrosis, and this microorganism can be highly transmissible. Pulsed-field gel electrophoresis is widely used to study the dynamics of strain spread in cystic fibrosis patients. The aim of this work was to perform pulsed-field gel electrophoresis-based molecular typing of B. cenocepacia isolates to evaluate the epidemiology of this species at our hospital. A total of 28 isolates from 23 cystic fibrosis patients were analyzed. Initially, we compared isolates obtained from the same patient at different periods of time. We then compared the pulsed-field gel electrophoresis profiles of 15 IIIA isolates, and in a third analysis, evaluated the genetic profile of 8 IIIB isolates from different patients. The pulsed-field gel electrophoresis profiles of isolates from the same patient indicated that they are genetically indistinguishable. Analysis of isolates from different patients revealed the presence of multiple clonal groups. These results do not indicate cross-transmission of a unique clone of B. cenocepacia among cystic fibrosis patients, although this has been observed in some patients. Our findings highlight the importance of adequate patient follow-up at cystic fibrosis centers and adherence to management and segregation measures in cystic fibrosis patients colonized with B. cenocepacia.

PCR to detect Mycobacterium tuberculosis in respiratory tract samples: evaluation of clinical data

Tuberculosis (TB) remains as an important public health problem worldwide. Therefore, the rapid detection of M. tuberculosis is of primary importance to effectively reduce transmission in patients. The aims of this study were to evaluate two in-house molecular tests: nested PCR (nPCR) and real-time PCR (rtPCR) to detect M. tuberculosis complex directly from clinical samples. The results were compared to the culture results and to the culture results plus clinical data of patients. The rtPCR and nPCR presented high sensitivity (Se) and specificity (Sp) (rtPCR 97·6% and 91·5%, nPCR 85·7% and 92·7%, respectively) compared to culture. When the results of the molecular tests were compared to the culture plus clinical data the Se and Sp were 90·2% and 97·3% for rtPCR and 80·4% and 98·6% for the nPCR, respectively. The results demonstrated that molecular assays of M. tuberculosis can provide a sensitive and rapid diagnostic of TB, and when used in addition to the clinical data of TB patients will help to improve the Sp of the diagnosis of pulmonary TB.

Evaluation of diagnostic tests for cytomegalovirus active infectionin renal transplant recipients

INTRODUCTION Cytomegalovirus (CMV) infection is a main viral infection after kidney transplantation. The diagnostic methods currently employed are pp65 antigenemia and nucleic acid amplification by polymerase chain reaction (PCR) and aim at detecting viral replication. OBJECTIVE The goal of this study was to evaluate and compare by both methods the incidence of CMV active infection in kidney transplant patients and to establishthe best clinical-laboratory correlation. METHODS Thirty sequential kidney transplant recipients were enrolled in a single center prospective cohort study. Peripheral blood samples were drawn from day 15 until the 6th month after transplantation and tested for CMV replication by pp65 antigenemia and quantitative PCR assays (qPCR). RESULTS Two hundred forty samples were analyzed and the incidence of active infection was similar by both methods. Time elapsed to the first positive test was almost identical but more samples tested positive by qPCR than by antigenemia in a behavior that was almost evenly distributed overtime. Agreement between tests was observed in 217 samples (90.4%; kappa = 0.529; p < 0.001) and in 25 patients the tests were concordant (83.3%; kappa = 0.667; p < 0.001). The evaluation of the diagnostic parameters for CMV replication revealed higher sensitivity for the qPCR test (82.1%) against antigenemia (59.0%). Quantitative PCR was also slightly more accurate than antigenemia. CONCLUSION Our data demonstrate that both methods are suitable and have almost equivalent accuracy for the detection of post-transplant cytomegalovirus replication. The choice for either test must take in consideration the demand, execution capability and cost-effectiveness at each institution.

Enhancing tuberculosis diagnosis by polymerase chain reaction: an experience at a tertiary hospital

Introduction: Tuberculosis (TB) persists as a severe global public health issue. The aim of the present study was to evaluate the performance of an in-house TB PCR (polymerase chain reaction) in sputum. Methods: DNA from sputum specimens were submitted to a nested-PCR protocol for the IS6110 region detection. PCR results were compared to those of the traditional methods for TB diagnosis, i.e., acid-fast bacilli (AFB) smear microscopy and culture. We analyzed sputum samples obtained from 133 patients. Results: A total of 48 (36%) cultures yielded indeterminate results due to contamination. This high contamination rate may be explained by the fact that samples from fibrocystic patients were included in this study. Additionally, other five samples were positive for nontuberculous mycobacteria (NTM). Therefore, it was possible to compare 80 patients for M. tuberculosis detection. We found 14 positive samples: five presented positive results in the three methods (5/14; 35.7%), two were positive in culture and PCR (2/14; 14.3%), one was positive in AFB and PCR (1/14; 7.1%), five were positive only in PCR (5/14; 35.7%) and 1 was positive only in culture (1/14; 7.1%). Thus, positivity rates for each technique were: 7.5% for AFB (6/80), 10% for culture (8/80) and 16.25% for PCR (13/80). Among the 48 patients who had indeterminate results in sputum culture, two samples were positive in PCR. Conclusion: Considering the limitations of the traditional methods, the use of PCR as a molecular technique could be advantageous for TB diagnosis.

Comparison of the performance of polymerase chain reaction and pp65 antigenemia for the detection of human cytomegalovirus in immunosuppressed patients

INTRODUCTION Human cytomegalovirus (HCMV) is often reactive in latently infected immunosuppressed patients. Accordingly, HCMV remains one of the most common infections following solid organ and hemopoietic stem cell transplantations, resulting in significant morbidity, graft loss and occasional mortality. The early diagnosis of HCMV disease is important in immunosuppressed patients, since in these individuals, preemptive treatment is useful. The objective of this study was to compare the performance of the in-house qualitative polymerase chain reaction (PCR) and pp65 antigenemia to HCMV infection in immunosuppressed patients in the Hospital de Clínicas of Porto Alegre (HCPA). METHODS A total of 216 blood samples collected between August 2006 and January 2007 were investigated. RESULTS Among the samples analyzed, 81 (37.5%) were HCMV-positive by PCR, while 48 (22.2%) were positive for antigenemia. Considering antigenemia as the gold standard, sensitivity, specificity, positive predictive values and negative predictive values for PCR were 87.5%, 76.8%, 51.8% and 95.5% respectively. CONCLUSIONS These results demonstrated that qualitative PCR has high sensitivity and negative predictive value (NPV). Consequently PCR is especially indicated for the initial diagnosis of HCMV infection. In the case of preemptive treatment strategy, identification of patients at high-risk for HCMV disease is fundamental and PCR can be useful tool.