Revisión sistemática de la exactitud diagnóstica de la gota gruesa en comparación con la reacción en cadena de la polimerasa para malaria asociada al embarazo, 2010-2022 / Systematic review of the diagnostic accuracy of thick smear compared to polymerase chain reaction for pregnancy-associated malaria, 2010-2022

RESUMEN Objetivos. Evaluar la exactitud de gota gruesa (GG) frente a la reacción en cadena de la polimerasa (PCR) cuantitativa para la malaria asociada al embarazo (MAE). Materiales y métodos. Se realizó una revisión sistemática de pruebas diagnósticas en nueve bases de datos. Se evaluó la calidad metodológica con QUADAS. Se estimó sensibilidad, especificidad, cociente de probabilidad positivo (CPP) y negativo (CPN), razón de odds diagnóstica (ORD) y área bajo la curva ROC. Se determinó la heterogeneidad con el estadístico Q de Der Simonian-Laird y la incertidumbre con el porcentaje de peso de cada estudio sobre el resultado global. Resultados. Se incluyeron diez estudios con 5691 gestantes, 1415 placentas y 84 neonatos. En los estudios con nPCR (PCR anidada) y qPCR (PCR cuantitativa) como estándar, los resultados de exactitud diagnóstica fueron estadísticamente similares, con sensibilidad muy baja (50 y 54%, respectivamente), alta especificidad (99% en ambos casos), alto CPP y deficiente CPN. Usando nPCR la OR diagnóstica fue 162 (IC95%=66-401) y el área bajo la curva ROC fue 95%, mientras que con qPCR fueron 231 (IC95%=27-1951) y 78%, respectivamente. Conclusiones. Mediante un protocolo exhaustivo se demostró el bajo desarrollo de investigaciones sobre la exactitud diagnóstica de la GG en MAE. Se demostró que la microscopía tiene un desempeño deficiente para el diagnóstico de infecciones asintomáticas o de baja parasitemia, lo que afianza la importancia de implementar otro tipo de técnicas en el seguimiento y control de las infecciones por malaria en las gestantes, con el fin de lograr el control y posible eliminación de la MAE.

ABSTRACT Objective. To evaluate the accuracy of thick smear (TS) versus quantitative polymerase chain reaction (PCR) for pregnancy-associated malaria (PAM). Materials and methods. We carried out a systematic review of diagnostic tests in nine databases. Methodological quality was evaluated with QUADAS. Sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR) and area under the ROC curve were estimated. Heterogeneity was determined with the Der Simonian-Laird Q method and uncertainty with the weighted percentage of each study on the overall result. Results. We included 10 studies with 5691 pregnant women, 1415 placentas and 84 neonates. In the studies with nested PCR (nPCR) and quantitative PCR (qPCR) as the standard, the diagnostic accuracy results were statistically similar, with very low sensitivity (50 and 54%, respectively), high specificity (99% in both cases), high PLR and poor NLR. When nPCR was used, the DOR was 162 (95%CI=66-401) and the area under the ROC curve was 95%, while with qPCR it was 231 (95%CI=27-1951) and 78%, respectively. Conclusions. We demonstrated that research on the diagnostic accuracy of TS in PAM is limited. Microscopy showed poor performance in the diagnosis of asymptomatic or low parasitemia infections, which reinforces the importance of implementing other types of techniques for the follow-up and control of malaria infections in pregnant women, in order to achieve the control and possible elimination of PAM.

Standardization of molecular techniques for the detection and characterization of intestinal protozoa and other pathogens in humans

Background: The intrinsic sensitivity limitations of basic parasitological methods, along with the particular biological characteristics of parasites, make these methods ineffective to differentiate morphologically indistinguishable species. Molecular detection and characterization techniques could be used to overcome these problems. The purpose of this work was to standardize molecular polymerase chain reaction (PCR) techniques, described in the literature, for the detection and molecular characterization of intestinal protozoa and other pathogens in humans. Methods: DNA was extracted from human or animal feces, previously washed or cultured in Boeck Drbohlav's Modified Medium. DNA extraction was performed with Machery-Nagel extraction kits. The standardization of the PCR, nested-PCR or RFLP techniques was carried out according to the literature. For each molecular technique performed, the sensitivity of the test was determined based on the minimun quantity required of DNA (sensitivity A) and the minimum quantity of life forms that the test detected (sensitivity B). Results: Sensitivity A was 10 fg for G. duodenalis, 12.5 pg for Entamoeba histolytica or Entamoeba dispar, 50 fg for Cryptosporidium spp., 225 pg for Cyclospora spp. and 800 fg or 8 fg for Blastocystis spp. after performing a 1780 bp PCR or 310 bp nested PCR, respectively. The sensitivity B was 100 cysts for G. duodenalis, 500 cysts for E. histolytica or E. dispar, 1000 oocysts for Cyclospora spp. and 3600 or four vegetatives forms for PCR or nested PCR of Blastocystis spp., respectively. Conclusions: The molecular detection of protozoa and chromist was achieved and the molecular characterization allowed the genotyping of some of the parasites such as Giardia duodenalis, Cryptosporidium spp., and Blastocystis spp. This study summarizes the molecular techniques for epidemiological studies in humans and animals, and helps in the investigation of their transmission sources in countries where intestinal parasites are a public health problem.(AU)

Directrices de Laboratorio para la Detección y el Diagnóstico de la Infección con el Virus COVID-19 / Laboratory Guidelines for the Detection and Diagnosis of COVID-19 Virus Infection

Los coronavirus son un grupo de virus ARN altamente diversos de la familia Coronaviridae que se dividen en 4 géneros: alfa, beta, gamma y delta, y que causan enfermedades de leves a graves en humanos y animales (1-3). Existen coronavirus humanos endémicos como los alfacoronavirus 229E y NL63 y los betacoronavirus OC43 y HKU1 que pueden causar enfermedades de tipo influenza o neumonía en humanos (1, 3). Sin embargo, dos coronavirus zoonóticos que causan enfermedades graves en humanos han emergido: el coronavirus del Síndrome respiratorio agudo grave (SARS-CoV) en 2002-2003 y el coronavirus del Síndrome respiratorio de Oriente Medio (MERS-CoV) (1-5). En enero de 2020, el agente etiológico responsable de un grupo de casos de neumonía grave en Wuhan, China, fue identificado como un nuevo betacoronavirus, distinto del SARS-CoV y MERS-CoV (6). El 11 de febrero de 2020, el Comité Internacional de Taxonomía de Virus (ICTV) anunció la denominación del virus como coronavirus del síndrome respiratorio agudo grave 2 (SARS-CoV-2) (7), mientras que, el mismo día, la OMS nombró la enfermedad como enfermedad por coronavirus COVID-19 (8). Para fines de comunicación, haremos referencia a este virus como "el virus responsable de COVID-19" o "el virus COVID-19". La secuencia genómica completa de este nuevo agente está disponible y se han desarrollado diferentes protocolos de detección (9). A la luz de la circulación actual de COVID-19 en la región de las Américas, la Organización Panamericana de la Salud / Organización Mundial de la Salud (OPS / OMS) recomienda a los Estados Miembros garantizar la identificación oportuna de casos sospechosos, la toma y el envío de muestras a los laboratorios de referencia, y la implementación de protocolos de detección molecular, según la capacidad del laboratorio.

Prevalence of Trichomonas vaginalis in Women Visiting 2 Obstetrics and Gynecology Clinics in Daegu, South Korea

This study explored epidemiological trends in trichomoniasis in Daegu, South Korea. Wet mount microscopy, PCR, and multiplex PCR were used to test for Trichomonas vaginalis in vaginal swab samples obtained from 621 women visiting 2 clinics in Daegu. Of the 621 women tested, microscopy detected T. vaginalis in 4 (0.6%) patients, PCR detected T. vaginalis in 19 (3.0%) patients, and multiplex PCR detected T. vaginalis in 12 (1.9%) patients. Testing via PCR demonstrated high sensitivity and high negative predictive value for T. vaginalis. Among the 19 women who tested positive for T. vaginalis according to PCR, 94.7% (18/19) reported vaginal signs and symptoms. Notably, more than 50% of T. vaginalis infections occurred in females younger than 30 years old, and 58% were unmarried. Multiplex PCR, which simultaneously detects pathogens from various sexually transmitted infections, revealed that 91.7% (11/12) of patients were infected with 2 or more pathogens. Mycoplasma hominis was the most prevalent co-infection pathogen with T. vaginalis, followed by Ureaplasma urealyticum and Chlamydia trachomatis. Our results indicate that PCR and multiplex PCR are the most sensitive tools for T. vaginalis diagnosis, rather than microscopy which has been routinely used to detect T. vaginalis infections in South Korea. Therefore, clinicians should take note of the high prevalence of T. vaginalis infections among adolescent and young women in order to prevent persistent infection and transmission of this disease.

Polymerase chain reaction detection of LeishmaniaDNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani

Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detectLeishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmaniagenus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.

Validation of GlobalFiler® PCR Amplification Kit and the STR Polymorphism / 法医学杂志

OBJECTIVE@#To test the technical parameters of GlobalFiler® PCR Amplification Kit for its application to forensic application value and to investigate the genetic polymorphisms.@*METHODS@#The validation was conducted in sensitivity, mixed samples, species specificity, adaptability, survivability, consistency, peak height balance and stability. The amplification and detection of the genomic DNA from 373 unrelated individuals from Beijing Han nationality were extracted by automation workstation.@*RESULTS@#Global-Filer® PCR Amplification Kit was adaptive to some mixed, degraded and inhibited samples. The power of sensitivity and adaptability and peak height balance showed well. The distributions of genotype frequencies for 21 STR loci in the population were all in accordance with Hardy-Weinberg equilibrium (P > 0.05). The PIC value of the 21 STR loci was among 0.536 to 0.940; the H value was among 0.558 to 0.933; the DP value was among 0.783 to 0.992; the PE value was among 0.243 to 0.874.@*CONCLUSION@#GlobalFiler® PCR Amplification Kit is suitable for criminal cases and DNA database in forensic practice. And 21 STR loci in Beijing Han nationality have high polymorphism, which have application value in forensic practice and population genetics.

Detection of Wuchereria bancrofti DNA in paired serum and urine samples using polymerase chain reaction-based systems

The Global Program for the Elimination of Lymphatic Filariasis (GPELF) aims to eliminate this disease by the year 2020. However, the development of more specific and sensitive tests is important for the success of the GPELF. The present study aimed to standardise polymerase chain reaction (PCR)-based systems for the diagnosis of filariasis in serum and urine. Twenty paired biological urine and serum samples from individuals already known to be positive for Wuchereria bancrofti were collected during the day. Conventional PCR and semi-nested PCR assays were optimised. The detection limit of the technique for purified W. bancrofti DNA extracted from adult worms was 10 fg for the internal systems (WbF/Wb2) and 0.1 fg by using semi-nested PCR. The specificity of the primers was confirmed experimentally by amplification of 1 ng of purified genomic DNA from other species of parasites. Evaluation of the paired urine and serum samples by the semi-nested PCR technique indicated only two of the 20 tested individuals were positive, whereas the simple internal PCR system (WbF/Wb2), which has highly promising performance, revealed that all the patients were positive using both samples. This study successfully demonstrated the possibility of using the PCR technique on urine for the diagnosis of W. bancrofti infection.

Parasite load and risk factors for poor outcome among children with visceral leishmaniasis. A cohort study in Belo Horizonte, Brazil, 2010-2011

Clinical and laboratory risk factors for death from visceral leishmaniasis (VL) are relatively known, but quantitative real-time polymerase chain reaction (qPCR) might assess the role of parasite load in determining clinical outcome. The aim of this study was to identify risk factors, including parasite load in peripheral blood, for VL poor outcome among children. This prospective cohort study evaluated children aged ≤ 12 years old with VL diagnosis at three times: pre-treatment (T0), during treatment (T1) and post-treatment (T2). Forty-eight patients were included and 16 (33.3%) met the criteria for poor outcome. Age ≤ 12 months [relative risk (RR) 3.51; 95% confidence interval (CI) 1.89-6.52], tachydyspnoea (RR 3.46; 95% CI 2.19-5.47), bacterial infection (RR 3.08; 95% CI 1.27-7.48), liver enlargement (RR 3.00; 95% CI 1.44-6.23) and low serum albumin (RR 7.00; 95% CI 1.80-27.24) were identified as risk factors. qPCR was positive in all patients at T0 and the parasite DNA was undetectable in 76.1% of them at T1 and in 90.7% at T2. There was no statistical association between parasite load at T0 and poor outcome.

Otimização de ferramentas moleculares baseadas em multiplex PCR para inclusão de controles de qualidade amostral no diagnóstico das leishmanioses / Development of molecular tools multiplex PCR-based for the inclusion of sample quality controls in the diagnosis of leishmaniasis

A detecção precoce das leishmanioses e a rápida instituição do tratamento são de suma importância para os indivíduos e comunidades afetadas, visto que pacientes contribuem para a manutenção do ciclo da doença. Diante das limitações apresentadas pelos métodos tradicionais de diagnóstico, a PCR tem se apresentado como ferramenta promissora para a detecção dos casos. No entanto, perdas de DNA durante o processo de purificação podem afetar mais significativamente o material genético dos parasitos, gerando resultados falso-negativos. Este estudo teve como objetivo desenvolver e avaliar dois protocolos de triplex PCR para investigar possíveis causas de negatividade no diagnóstico molecular das formas visceral (LV) e tegumentar (LT) das leishmanioses. Pares de primers para detecção de um controle interno (gene G3PD) e dois controles externos (DNA genômico de M. pachydermatis e plasmídeo comercial pUC18 foram adaptados a protocolos de PCR convencionais validados para a detecção de L. infantum e L.(V.) braziliensis e duas reações triplex foram otimizadas. Dados de sensibilidade (S), especificidade (E) e eficiência (e) dos novos sistemas foram calculados em 186 amostras de sangue coletadas de cães em áreas endêmicas, utilizando como referência protocolos de PCR e PCR em tempo real (qPCR) consagrados na literatura. A concordância entre os novos testes e os testes de referência foi determinada pelo cálculo do índice de Kappa. A tríplex PCR para o diagnóstico da LV mostrou S = 78.68 por cento, E= 85.29 por cento e e= 81.05 por cento com boa concordância (K = 0.60, p< 0.0001) com o conjunto de resultados PCR/qPCR. Para o diagnóstico da LT observou-se S = 97.29 por cento, E = 79.16 por cento, e = 90.16 por cento, com K = 0.78, (p<0.0001) indicando excelente nível de concordância entre os testes. As novas ferramentas apresentadas podem ser aplicadas para aumentar a acurácia no diagnóstico das leishmanioses, contribuindo para a rápida implementação do tratamento e, reduzindo a longo prazo os índices de mortalidade e morbidade das leishmanioses.

Application of HTB-SiHa Cells Transfected with a Recombinant Plasmid for External Quality Assessment of Chlamydia trachomatis PCR

BACKGROUND: The participation of laboratories in external quality assessment (EQA) programs is required for the quality assurance of nucleic acid amplification of Chlamydia trachomatis. This study aimed to construct a new quality control (QC) material applicated in EQA of C. trachomatis PCR. METHODS: A QC material-HTB-SiHa cells transfected with a recombinant plasmid containing the cryptic plasmid sequence-was constructed for C. trachomatis PCR detection, and four different panels, each consisting of 4 positive samples with serial dilution of the constructed QC material and 1 negative sample, were distributed by the National Center for Clinical Laboratories among four groups of 275, 268, 317, and 304 participants across China from 2011 through 2012. A total of eight commercial kits were used for C. trachomatis PCR detection in participants. RESULTS: Nine laboratories reported false-positive results (0.9%). As the series dilution increased, the correct reporting of the data sets decreased; the lowest correct rate was 96.3% in the weakest positive samples (104 copies/mL). Eight laboratories reported false-positive results, and 42 laboratories reported false-negative results in the EQA detection of C. trachomatis. No significant differences were observed in the detection of the constructed C. trachomatis positive samples (97.9%, 98.5%, 100%, 98.5%; P=0.36) and negative samples (100%, 99.0%, 100%, 99.0%; P=0.764) using four commercial kits commonly used in China. CONCLUSIONS: The results of the EQA study indicated that the constructed material provides a noninfectious, stable control material with sufficient volume for PCR detection of C. trachomatis.

Performance evaluation of the HepB Typer-Entecavir kit for detection of entecavir resistance mutations in chronic hepatitis B

BACKGROUND/AIMS: Molecular diagnostic methods have enabled the rapid diagnosis of drug-resistant mutations in hepatitis B virus (HBV) and have reduced both unnecessary therapeutic interventions and medical costs. In this study we evaluated the analytical and clinical performances of the HepB Typer-Entecavir kit (GeneMatrix, Korea) in detecting entecavir-resistance-associated mutations. METHODS: The HepB Typer-Entecavir kit was evaluated for its limit of detection, interference, cross-reactivity, and precision using HBV reference standards made by diluting high-titer viral stocks in HBV-negative human serum. The performance of the HepB Typer-Entecavir kit for detecting mutations related to entecavir resistance was compared with direct sequencing for 396 clinical samples from 108 patients. RESULTS: Using the reference standards, the detection limit of the HepB Typer-Entecavir kit was found to be as low as 500 copies/mL. No cross-reactivity was observed, and elevated levels of various interfering substances did not adversely affect its analytical performance. The precision test conducted by repetitive analysis of 2,400 replicates with reference standards at various concentrations showed 99.9% agreement (2398/2400). The overall concordance rate between the HepB Typer-Entecavir kit and direct sequencing assays in 396 clinical samples was 99.5%. CONCLUSIONS: The HepB Typer-Entecavir kit showed high reliability and precision, and comparable sensitivity and specificity for detecting mutant virus populations in reference and clinical samples in comparison with direct sequencing. Therefore, this assay would be clinically useful in the diagnosis of entecavir-resistance-associated mutations in chronic hepatitis B.

Standardization of fungal polymerase chain reaction for the early diagnosis of invasive fungal infection.

Background: An early initiation of antifungal therapy in invasive fungal infections (IFIs) is critical in reducing the high mortality rate. Current diagnosis of fungal infection relies on microscopy, culture, antigen, antibody specific tests and histological diagnosis. However, these tests either lack sensitivity or specificity. There is thus the need for a rapid, specific and accurate diagnostic method. Objective: The aim of our study was to establish PCR for the rapid detection of Candida and Aspergillus species in clinical specimens with improved sensitivity and specificity. Materials and Methods: A total of 71 proven cases of IFI (confirmed by culture) were collected. A total of 15 healthy, 15 patients suffering from bacterial sepsis and 15 patients with HIV, HBV viral infections were included as controls. Clinical specimens were subjected to a standardized nested amplification to produce Round I (504 bp) and Round II (150 bp) amplicons. Restriction digestion was performed on these products for further identification. Results: Analytical sensitivity was determined using 10 6 -10 CFU/ml of cell suspension. The lower detection limit of the assay was 10 CFU/ml of blood. This test was 100% sensitive and specific with a positive predictive value of 100% and a negative predictive value of 96.7%. Conclusion: The assay was found to be effective for the rapid detection of Candida and Aspergillus in clinical specimens.

Comparison of Diagnostic Performance of Three Real-Time PCR Kits for Detecting Mycobacterium Species

PURPOSE: PCR is widely used for rapidly and accurately detecting Mycobacterium Species. The purpose of this study was to assess the diagnostic performance of three real-time PCR kits and evaluate the concordance with two older PCR methods. MATERIALS AND METHODS: Using 128 samples, the five PCR methods were assessed, including an in-house PCR protocol, the COBAS Amplicor MTB, the COBAS TaqMan MTB, the AdvanSure TB/NTM real-time PCR, and the Real-Q M. tuberculosis kit. The discrepant results were further examined by DNA sequencing and using the AdvanSure Mycobacteria Genotyping Chip for complete analysis. RESULTS: For Mycobacterium tuberculosis (MTB) detection, all five kits showed 100% matching results (positive; N = 11 and negative; N = 80). In non-tuberculous mycobacterium (NTM) discrimination, the AdvanSure yielded two true-positive outcomes from M. intracellulare and one false positive outcome, while the Real-Q resulted in one true-positive outcome and one false negative outcome for each case and another false negative result using the provided DNA samples. CONCLUSION: Real-time PCR, yielded results that were comparable to those of the older PCR methods for detecting MTB. However, there were disagreements among the applied kits in regard to the sample test results for detecting NTM. Therefore, we recommend that additional confirmatory measures such as DNA sequencing should be implemented in such cases, and further research with using a larger numbers of samples is warranted to improve the detection of NTM.

Aplicación de las pruebas de PCR convencional simple y múltiple para la identificación de aislamientos de Leptospira spp. en Colombia / Application of conventional and multiplex PCR assays for identification of isolates of Leptospira spp. in Colombia

Debido a las dificultadas asociadas con la identificación serológica de aislamientos de Leptospira ssp, se genera gran interés en la pruebas moleculares por su poder discriminatorio, reproducibilidad y fácil interpretación. Objetivo. Aplicar y validar la prueba de PCR convencional, usando dos pares de iniciadores descritos previamente y dirigidos a los genes lipL32 (PCR simple) y secY/flaB (PCR múltiple), con el fin de evaluar su aplicación para identificar especies patógenas y saprófitas de Leptospira spp. Materiales y métodos. Para la estandarización de las pruebas de PCR se usó 22 cepas de referencia internacional y 12 aislamientos colombianos. Se determinó el nivel de detección de cada pareja de iniciadores, su especificidad frente a otros microorganismos causantes de enfermedades endémicas en Colombia y su capacidad de identificar especies dentro del grupo de Leptospira. Resultados. El límite de detección de la PCR simple lipL32 fue una dilución 1:10000 y para la PCR múltiple secY/flaB fue una dilución 1:100 para el gen secY y 1:1000 para flaB. La especificidad de todos los iniciadores fue de 100 por ciento. La PCR simple lipL32, mostró amplificado específico para 21/22 cepas de referencia mientras que la PCR multiple secY/flaB lo fue para 18/22 cepas. De los 12 aislamientos colombianos, siete fueron positivos por PCR lipL32 y seis lo fueron por PCR secY/flaB. Conclusiones. Los resultados más consistentes fueron obtenidos con la PCR simple lipL32 tanto en límite de detección, especificidad y utilidad para la identificación de Leptospira spp, por lo que esta prueba es aplicable a la identificación molecular de aislamientos patógenos de Leptospira spp de diversas fuentes.

Serological identification of Leptospira ssp isolates is difficult to achieve. Thus, molecular testing may be of great interest thanks to its high discrimination power, reproducibility and easy interpretation. Objective. To implement and validate conventional and multiplex PCR methods (using primers directed against lipl32 and secY/flaB genes, respectively). To assess the capacity of PCR methods to identify pathogenic and saprophytic species of Leptospira ssp. Material and methods. 22 international reference strains and 12 colombian isolates were used. DNA was extracted with a commercial kit (Wizard). Specificity and sensitivity of both PCR methods were evaluated. Results. The maximum dilution of DNA samples allowing the detection of Leptospira ssp was determined to be 1:10000 for the PCR lipL32 and 1:100/1:1000 for the multiplex PCR secY/flaB. Both PCR didn’t detect DNA from microorganisms unrelated to Leptospira ssp. The lipL32 PCR specifically amplified a 423bp fragment from all pathogenic Leptospira reference strains, while the secY/flaB PCR amplified both 285bp (secY) and 793bp (flaB) fragments from 18 reference strains. The lipL32 PCR detected 7/12 colombian isolates, while secY/flaB PCR detected both secY and flaB genes from 6/12 isolates. Conclusions. Best results were obtained with the lipL32 PCR, which displayed a better sensitivity and a better capacity to detect different strains than the multiplex PCR. The secY primers showed a poor specificity to pathogenic species and a poor sensitivity. Thus, lipL32 primers show high potential for molecular diagnosis of Leptospira spp in clinical and environmental samples.

Polymerase chain reaction-Based clonality analysis of cutaneous B-cell lymphoproliferative processes

Introduction: The differential diagnosis of B-cell lymphoproliferative processes remains a challenge for pathologists, dermatologists and oncologists, despite advances in histology, immunohistochemistry and molecular biology. Objective: Evaluate aid and limitations of clonality analysis in the diagnosis of primary cutaneous B-cell lymphomas and B-cell pseudolymphomas. Methods: This study included 29 cases of B-cell lymphoproliferative processes classified as primary cutaneous B-cell lymphomas (13), B-cell pseudolymphomas (6) and inconclusive cases (10) using histology and immunohistochemistry. The clonality analysis was performed by polymerase chain reaction analysis of immunoglobulin light chain and heavy chain rearrangements. Results: DNA quality was shown to be generally poor; eight samples were inadequate for polymerase chain reaction analysis. The results showed monoclonality in eight of the primary cutaneous B-cell lymphomas and polyclonality in four of the B-cell pseudolymphomas. In addition, monoclonality was shown in two of the inconclusive cases by histology and immunohistochemistry, demonstrating the utility of polymerase chain reaction as an ancillary diagnostic tool for primary cutaneous B-cell lymphomas. Discussion: The low quality DNA extracted from these cases demanded the use of an IgH protocol that yielded small fragments and IgK. Both methods used together improved detection. Conclusion: Use of the two protocols, immunoglobulin heavy chain FR3-trad and immunoglobulin light chain-Kappa Biomed protocols for clonality analysis improved diagnostic accuracy.

Desempenho da técnica nested PCR na detecção específica do complexo Mycobacterium tuberculosis em amostras sanguíneas de pacientes pediátricos / Performance of nested PCR in the specific detection of Mycobacterium tuberculosis complex in blood samples of pediatric patients

Objetivo: Avaliar o desempenho da técnica nested PCR (nPCR) para detectar o complexo Mycobacterium tuberculosis em amostras de sangue de pacientes com suspeita de TB para sua possível utilização como uma ferramenta auxiliar no diagnóstico laboratorial da doença em crianças. Métodos: Detecção do complexo M. tuberculosis em amostras de sangue usando como alvo a sequência de inserção IS6110 do DNA genômico do bacilo. Foram avaliados 120 pacientes, menores de 15 anos de idade, de ambos os sexos, provenientes de hospitais públicos do Recife (PE), no período entre janeiro de 2003 e agosto de 2005. O diagnóstico de TB foi realizado pelo médico assistente do serviço de saúde de acordo com os critérios da Sociedade Torácica Americana. A nPCR amplificou um fragmento de 123 pb com oligonucleotídeos externos (IS1/IS2) e, na reação subsequente, com oligonucleotídeos internos (IS3/IS4), gerando um amplicon de 81 pb. Resultados: A TB ativa ou latente esteve presente em 65 pacientes, foi descartada em 28 suspeitos e 27 não tinham a doença (controles). A sensibilidade da nPCR foi de 26,15%, sendo significativamente maior na forma extrapulmonar (55,56%) em relação à pulmonar (18,18%), e a especificidade foi de 92,73%. Conclusões: Diante das dificuldades diagnósticas da TB infantil e do baixo número de casos estudados,a nPCR em sangue demonstrou ser uma técnica rápida e específica, mas com baixa sensibilidade. Para saber a suareal utilidade no diagnóstico de formas paucibacilares, sobretudo as extrapulmonares, novas pesquisas devem ser desenvolvidas com uma casuística maior de crianças e com outros espécimes biológicos além do sangue.

Objective: To evaluate the performance of nested PCR (nPCR) in detecting the Mycobacterium tuberculosis complex in blood samples of patients suspected of having TB, in order to determine its potential for use as an auxiliary tool in the laboratory diagnosis of TB in children. Methods: Detection of the M. tuberculosis complex in blood samples using as a target the insertion sequence IS6110 of the genomic DNA of the bacillus. Blood samples of 120 patients were evaluated. All of the patients were under 15 years of age at the time of their treatment at public hospitals in the city of Recife, Brazil (between January of 2003 and August of 2005). Attending physicians at the hospitals diagnosed TB based on the criteria recommended by the American Thoracic Society. The nPCRamplified a 123-bp fragment with outer oligonucleotides (IS1/IS2) and, in the subsequent reaction, using inner oligonucleotides (IS3/IS4), generating an 81-bp amplicon. Results: Active or latent TB was found in 65 patients,TB was ruled out in 28 suspected cases, and 27 patients were TB-free (controls). The sensitivity of nPCR was26.15% and was significantly higher for the extrapulmonary form of the disease (55.56%) than for the pulmonary form (18.18%). The specificity was 92.73%. Conclusions: Despite the difficulties in diagnosing TB in children and the low number of cases evaluated in the present study, nPCR in blood samples proved to be a rapid and specifictechnique, albeit one with low sensitivity. In order to establish its true usefulness in the diagnosis of paucibacillary forms, especially extrapulmonary TB, further studies need to be carried out with a larger sample of children and analyzing biological specimens other than blood.

Significant performance variation among PCR systems in diagnosing congenital toxoplasmosis in São Paulo, Brazil: analysis of 467 amniotic fluid samples

INTRODUCTION: Performance variation among PCR systems in detecting Toxoplasma gondii has been extensively reported and associated with target genes, primer composition, amplification parameters, treatment during pregnancy, host genetic susceptibility and genotypes of different parasites according to geographical characteristics. PATIENTS: A total of 467 amniotic fluid samples from T. gondii IgM- and IgG-positive Brazilian pregnant women being treated for 1 to 6 weeks at the time of amniocentesis (gestational ages of 14 to 25 weeks). METHODS: One nested-B1-PCR and three one-round amplification systems targeted to rDNA, AF146527 and the B1 gene were employed. RESULTS: Of the 467 samples, 189 (40.47 percent) were positive for one-round amplifications: 120 (63.49 percent) for the B1 gene, 24 (12.69 percent) for AF146527, 45 (23.80 percent) for both AF146527 and the B1 gene, and none for rDNA. Fifty previously negative one-round PCR samples were chosen by computer-assisted randomization analysis and re-tested (nested-B1-PCR), during which nine additional cases were detected (9/50 or 18 percent). DISCUSSION: The B1 gene PCR was far more sensitive than the AF146527 PCR, and the rDNA PCR was the least effective even though the rDNA had the most repetitive sequence. Considering that the four amplification systems were equally affected by treatment, that the amplification conditions were optimized for the target genes and that most of the primers have already been reported, it is plausible that the striking differences found among PCR performances could be associated with genetic diversity in patients and/or with different Toxoplasma gondii genotypes occurring in Brazil. CONCLUSION: The use of PCR for the diagnosis of fetal Toxoplasma infections in Brazil should be targeted to the B1 gene when only one gene can be amplified, preferably by nested amplification with primers B22/B23.

Genotipos de Staphylococcus aureus con fenotipo meticilino resistente, aislados de pacientes del Hospital Base de Valdivia / Genotypes of Staphylococcus aureus strains with methicillin resistant phenotype

Background: Methicillin resistant strains of Staphylococcus aureus (MRSA) are an important cause of nosocomial infections. Aim: To determine the genotypes of MRSA strains. Material and methods: Fifty five strains of MRSA, isolated from patients hospitalized in Hospital Base Valdivia, were studied. The phenotype was determined through MicroScan® in all strains and by minimum inhibitory concentration (MIC) in 41. The genotype of the strains was analyzed by a duplex polymerase chain reaction (PCR) of the mecA gene, amplifying eight hypervariable DNA regions associated to such gene. Results: According to MIC, 88 percent of strains had a pattern of resistance against multiple antimicrobial (penicillin, ampicillin, cephradine, gentamycin, ciprofloxacin, íincomycin and erythromycin). Vancomicin resistan strains were not detected. Only 53 strains (96 percent) had at least one of the eight hypervariable regions and were classified as MRSA. Genotypic patterns types 15 were the most commonly detected in 38 percent and 34 percent of strains, respectively. MicroScan® erroneously classified five strains in an incorrect phenotype, according to results obtained with duplex PCR. MIC results did not differ from those of duplex PCR. Conclusions: Duplex- PCR is a useful tool to detect hyper variable regions associated to mecA gene.

Estandarización de una técnica de amplificación genómica (PCR) para el diagnóstico de dermatofitos / Standardization of a technique of genomic amplification (pcr) for diagnose of dermatophites

Los dermatofitos constituyen un grupo bien definido de hongos filamentosos, taxonómicamente relacionados, que invaden y degradan tejidos queratinizados (piel, pelo y uñas) en humanos y otras especies y originan una variedad de lesiones clínicas llamadas dermatofitosis o tineas (tiñas). Los agentes etiológicos involucrados se clasifican en tres géneros: Epidermophyton, Microsporum y Trichophyton. En Chile, los agentes aislados corresponden a M. Canis, T. Rubrum, T. Mentagrophytes, E. Floccosum y ocasionalmente se han descrito aislamientos de T. Schoenleinii, T. Verrucosum y M. Gypseum. Este estudio presenta la estandarización de una técnica de Reacción de Polimerasa en cadena (PCR) cuyo objetivo es mejorar el diagnóstico de estos microorganismos en el laboratorio de Micología. Se utilizaron partidores para hongos. 30 cepas de dermatofitos aisladas de pacientes con patología activa, y 30 muestras fisiológicas positivas y negativas (escamas de piel, uñas y pelos) atendidos en el Hospital del Trabajador de Santiago. Se obtuvo una concordancia de 93,3 por ciento con los resultados obtenidos por cultivo, además de un 100 por ciento de concordancia entre los resultados por PCR y los de las técnicas tradicionales de diagnóstico. Como conclusión, nuestros resultados sugieren que la técnica de PCR es un método rápido, sensible y especifico para la identificación de este grupo de hongos a partir de muestras provenientes de cultivos micológicos y de muestras clínicas superficiales.