Assessment of conventional PCR and real-time PCR compared to the gold standard method for screening Streptococcus agalactiae in pregnant women

ABSTRACT Group B Streptococcus is a causative agent of invasive neonatal infections. Maternal colonization by Streptococcus agalactiae is a necessary condition for vertical transmission, with efficient screening of pregnant women playing an essential role in the prevention of neonatal infections. In this study, we aimed to compare the performance of conventional polymerase chain reaction and real-time PCR assays as screening methods for S. agalactiae in pregnant women against the microbiological culture method considered as the gold-standard. A total of 130 samples from pregnant women were analyzed for sensitivity, specificity, positive predictive value, and negative predictive value. Statistical analysis was performed using the SPSS software, version 20.0. The verified colonization rate was 3.8% with the gold-standard, 17.7% with conventional PCR assay, and 29.2% with the real-time PCR test. The trials with conventional PCR and real-time PCR had a sensitivity of 100% and a specificity of 85.6% and 73.6%, respectively. The real-time PCR assay had a better performance compared to the gold-standard and a greater detection rate of colonization by S. agalactiae compared to conventional PCR assay. With its quick results, it would be suitable for using in routine screenings, contributing to the optimization of preventive approaches to neonatal S. agalactiae infection.

Optimization of one-step real-time PCR for the X-tail target of HCV as a diagnostic test

Infection with hepatitis C virus (HCV) is a global public health issue. The bloodborne nature of HCV transmission poses a substantial risk to healthcare workers, due to occupational exposure to needlestick injuries and blood and other body fluids containing the virus. Undiagnosed HCV infection, including in healthcare workers, represents a growing problem worldwide as the infected population ages, and HCV-related mortality and morbidity is expected to rise substantially over the coming decades. Consequently, diagnostic tests for HCV play an important role in this scenario. The aim of this study was to standardize a one-step RT-PCR assay for detection of HCV. The test demonstrated reproducibility, sensibility (100%), and the limit of detection was set at 100IU/mL. Our study indicates that this assay can be used as a diagnostic tool to follow up healthcare workers after occupational exposure

Minimum detection limit of an in-house nested-PCR assay for herpes simplex virus and varicella zoster virus

Introduction Herpes simplex virus (HSV) and varicella zoster virus (VZV) are responsible for a variety of human diseases, including central nervous system diseases. The use of polymerase chain reaction (PCR) techniques on cerebrospinal fluid samples has allowed the detection of viral DNA with high sensitivity and specificity. Methods Serial dilutions of quantified commercial controls of each virus were subjected to an in-house nested-PCR technique. Results The minimum detection limits for HSV and VZV were 5 and 10 copies/µL, respectively. Conclusions The detection limit of nested-PCR for HSV and VZV in this study was similar to the limits found in previous studies. .

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 percent) positive results. Sensitivity and specificity for PCR were 100 percent and 86.88 percent, 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.

Comparison of the performance of polymerase chain reaction and pp65 antigenemia for the detection of human cytomegalovirus in immunosuppressed patients / Comparação de métodos para detecção de infecção por citomegalovírus em pacientes imunossuprimidos

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 percent) were HCMV-positive by PCR, while 48 (22.2 percent) were positive for antigenemia. Considering antigenemia as the gold standard, sensitivity, specificity, positive predictive values and negative predictive values for PCR were 87.5 percent, 76.8 percent, 51.8 percent and 95.5 percent 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.

INTRODUÇÃO: O citomegalovírus humano (HCMV), causador de infecção latente, reativa com frequência em pacientes imunossuprimidos. Portanto, o HCMV permanece uma das infecções mais comuns após transplantes de órgãos sólidos e de células hematopoiéticas resultando em significativa morbidade, perda do enxerto e ocasional mortalidade. Assim, o diagnóstico precoce para uma terapia preventiva é de grande importância. Este estudo visa comparar o desempenho dos métodos PCR qualitativo in-house e antigenemia pp65 para o diagnóstico de infecção por CMV em pacientes imunossuprimidos do Hospital de Clínicas de Porto Alegre. MÉTODOS: O estudo foi realizado em 216 amostras de sangue total (EDTA) coletadas de 85 pacientes, entre agosto de 2006 e janeiro de 2007. RESULTADOS: Dentre as 216 amostras analisadas, 81 (37,5 por cento) amostras apresentaram resultados positivos na PCR, enquanto 48 (22,2 por cento) apresentaram resultados positivos na antigenemia. A sensibilidade, especificidade, valor preditivo positivo e valor preditivo negativo para a PCR, considerando antigenemia como padrão foram 87,5 por cento, 76,8 por cento, 51,8 por cento e 95,5 por cento, respectivamente. CONCLUSÕES: Estes resultados demonstraram que a PCR tem alta sensibilidade e valor preditivo negativo. Consequentemente PCR é especialmente indicada para o diagnóstico inicial de infecção por HCMV. No caso da estratégia de terapia preventiva, a identificação de pacientes com alto risco para a doença por HCMV é fundamental e a PCR pode ser uma ferramenta útil.

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 percent of staphylococci were positive for cMLS B; 3.3 percent for iMLS B and 3.3 percent for MS B. One or more erm genes were present in 50.1 percent 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 percent, 17.1 percent and 0.66 percent respectively, and constitutive resistance was the most frequent as compared to the other two phenotypes.

Diagnosis of disseminated toxoplasmosis by polymerase chain reaction in bronchoalveolar lavage fluid of a patient with aids / Diagnóstico de toxoplasmose disseminada através de técnica de reação em cadeia da polimerase realizada no lavado bronco-alveolar de paciente com sida

Pulmonary toxoplasmosis is a challenging diagnosis in immunosuppressed patients with nonspecific clinical picture and radiologic findings. We present a case of pneumonia due to Toxoplasma gondii diagnosed by polymerase chain reaction (PCR) in the bronchoalveolar lavage (BAL) fluid of a patient with acquired immunodeficiency syndrome (AIDS). Coinfection with Pneumocystis jirovecii was found in the same specimen. Direct examination and culture for bacteria, mycobacteria and other fungus were negative. Despite the intensive management, respiratory compromise evolved rapidly, with the need for ventilatory support. Acute respiratory distress syndrome developed, and the patient died of multiple organ failure. This case illustrates that a high index of suspicion is necessary for diagnosis of pulmonary toxoplasmosis, a potentially fatal condition. Due to high diagnostic performance, PCR in BAL fluid should be included in the evaluation of immunosuppressed patients with nonspecific pulmonary diseases.

O diagnóstico de toxoplasmose pulmonar em pacientes imunossuprimidos é difícil, devido ao quadro clínico e aos achados radiológicos inespecíficos. Neste artigo, relatamos o caso de uma paciente com síndrome da imunodeficiência adquirida (SIDA), que apresentou pneumonia por Toxoplasma gondii diagnosticada através de reação em cadeia da polimerase (PCR) no lavado bronco-alveolar (LBA). A paciente apresentava co-infecção com Pneumocystis jirovecii. Os demais exames microbiológicos, como bacterioscópico, cultural para bactérias, micobactérias e fungos, foram negativos. Apesar do manejo intensivo, a paciente evoluiu com síndrome do desconforto respiratório agudo e óbito por falência múltipla dos órgãos. Este caso demonstra que um alto índice de suspeita clínica é necessário para o diagnóstico de pneumonia por Toxoplasma gondii. Devido ao seu desempenho diagnóstico, o PCR para Toxoplasma gondii no LBA deve ser incluído na avaliação de pacientes imunossuprimidos com quadros pulmonares inespecíficos.