Multiple-clone infections of Plasmodium vivax: definition of a panel of markers for molecular epidemiology.

BACKGROUND: Plasmodium vivax infections commonly contain multiple genetically distinct parasite clones. The detection of multiple-clone infections depends on several factors, such as the accuracy of the genotyping method, and the type and number of the molecular markers analysed. Characterizing the multiplicity of infection has broad implications that range from population genetic studies of the parasite to malaria treatment and control. This study compared and evaluated the efficiency of neutral and non-neutral markers that are widely used in studies of molecular epidemiology to detect the multiplicity of P. vivax infection. METHODS: The performance of six markers was evaluated using 11 mixtures of DNA with well-defined proportions of two different parasite genotypes for each marker. These mixtures were generated by mixing cloned PCR products or patient-derived genomic DNA. In addition, 51 samples of natural infections from the Brazil were genotyped for all markers. The PCR-capillary electrophoresis-based method was used to permit direct comparisons among the markers. The criteria for differentiating minor peaks from artifacts were also evaluated. RESULTS: The analysis of DNA mixtures showed that the tandem repeat MN21 and the polymorphic blocks 2 (msp1B2) and 10 (msp1B10) of merozoite surface protein-1 allowed for the estimation of the expected ratio of both alleles in the majority of preparations. Nevertheless, msp1B2 was not able to detect the majority of multiple-clone infections in field samples; it identified only 6 % of these infections. The merozoite surface protein-3 alpha and microsatellites (PvMS6 and PvMS7) did not accurately estimate the relative clonal proportions in artificial mixtures, but the microsatellites performed well in detecting natural multiple-clone infections. Notably, the use of a less stringent criterion to score rare alleles significantly increased the sensitivity of the detection of multi-clonal infections. CONCLUSIONS: Depending on the type of marker used, a considerable amplification bias was observed, which may have serious implications for the characterization of the complexity of a P. vivax infection. Based on the performance of markers in artificial mixtures of DNA and natural infections, a minimum panel of four genetic markers (PvMS6, PvMS7, MN21, and msp1B10) was defined, and these markers are highly informative regarding the genetic variability of P. vivax populations.

Resistencia a los antirretrovirales: Informe preliminar del capítulo de Guatemala, del estudio de epidemiología molecular del VIH en la región de mesoamerica / Antiretroviral resistance: initial report of Guatemala chapter, the study of molecular epidemiology of HIV in the region of Mesoamerica

El uso más extendido de los fármacos antirretrovirales ha traído como consecuencia la transmisión de variantes virales con mutaciones de resistencia que se pueden mantener en individuos sin tratamiento antirretroviral. La frecuencia de estas mutaciones de resistencia transmitida es relativamente alta en países desarrollados, muchas veces con tendencias de aumento. En países en vías de desarrollo, en los que la terapia antirretroviral (ARV) se introdujo posteriormente, las frecuencias de resistencia primaria tienden a ser menores, probablemente porque su uso se encuentra basado en una disponibilidad relativamente limitada...

Establishment and operation of a biorepository for molecular epidemiologic studies in Costa Rica.

BACKGROUND: The Proyecto Epidemiológico Guanacaste (PEG) has conducted several large studies related to human papillomavirus (HPV) and cervical cancer in Guanacaste, Costa Rica in a long-standing collaboration with the U.S. National Cancer Institute. To improve molecular epidemiology efforts and save costs, we have gradually transferred technology to Costa Rica, culminating in state-of-the-art laboratories and a biorepository to support a phase III clinical trial investigating the efficacy of HPV 16/18 vaccine. OBJECTIVE: Here, we describe the rationale and lessons learned in transferring molecular epidemiologic and biorepository technology to a developing country. RESULTS: At the outset of the PEG in the early 1990s, we shipped all specimens to repositories and laboratories in the United States, which created multiple problems. Since then, by intensive personal interactions between experts from the United States and Costa Rica, we have successfully transferred liquid-based cytology, HPV DNA testing and serology, chlamydia and gonorrhea testing, PCR-safe tissue processing, and viable cryopreservation. To accommodate the vaccine trial, a state-of-the-art repository opened in mid-2004. Approximately 15,000 to 50,000 samples are housed in the repository on any given day, and >500,000 specimens have been shipped, many using a custom-made dry shipper that permits exporting >20,000 specimens at a time. Quality control of shipments received by the NCI biorepository has revealed an error rate of <0.2%. Recently, the PEG repository has incorporated other activities; for example, large-scale aliquotting and long-term, cost-efficient storage of frozen specimens returned from the United States. Using Internet-based specimen tracking software has proven to be efficient even across borders. CONCLUSION: For long-standing collaborations, it makes sense to transfer the molecular epidemiology expertise toward the source of specimens. The successes of the PEG molecular epidemiology laboratories and biorepository prove that the physical and informatics infrastructures of a modern biorepository can be transferred to a resource-limited and weather-challenged region. Technology transfer is an important and feasible goal of international collaborations.

Variation in Trypanosoma cruzi clonal composition detected in blood patients and xenodiagnosis triatomines: implications in the molecular epidemiology of Chile.

To identify Trypanosoma cruzi clones from chronically infected individuals, they were transferred to triatomines by the xenodiagnosis test (XD) with Triatoma infestans. Polymerase chain reaction (PCR) and hybridization assays were performed to detect minicircle DNA in human blood samples and triatomine feces, using probes to determine the T. cruzi clones present. T. cruzi clone 19 (TcI) resulted the most prevalent in humans, with a frequency of 0.70 compared with a frequency of 0.53 in triatomines. T. cruzi clone 39 (TcIId) was the most prevalent in T. infestans, with a frequency of 0.65 compared with 0.33 in humans. The T. cruzi clone 43 (TcIIe) was not detected in blood samples; nevertheless, it was present at a rate of 0.17 in T. infestans feces. In conclusion, the T. cruzi clones are associated to each host, suggesting that selective amplification of clones occurs in human and triatomines.

Identificación de factores de riesgo para tuberculosis mediante epidemiologia molecular en el Departamento de La Paz, Bolivia, diciembre de 1998 a mayo de 2000 / Identification of risk factors for tuberculosis throgh molecular epidemiology in the Departament of La Paz, Bolivia, december 1998 to may 2000

Tomando en cuenta la situación de la tuberculosis (TB) en Bolivia y en La Paz y con el afán de poder hallar soluciones al problema de la salud pública que representa, se pretende implementar un estudio de epidemiologia molecular con el empleo de la técnica llamada reacción en cadena de la Polimerasa de doble elemento repetitivo (DRE-PCR). tomando en cuenta las ventajas que proporcionael empleo del DRE-PCR para la subtipificación de capas de M. tuberculosis y aplicando epidemiologia molecular con un modelo de investigación de casos y controles, se pretende identificar los factores de riesgo principales que son responsables de la diseminación de la TB en el departamento de La Paz...

Molecular diversity of live-attenuated prototypic vaccine strains and clinical isolates of Staphylococcus aureus.

Bovine mastitis Staphylococcus aureus isolates and prototypic live-attenuated vaccine strains were analyzed by SmaI pulsed-field gel electrophoresis (PFGE) typing and automated ribotyping. The discriminatory index of these methods was 0.91 and 0.69, respectively. SmaI PFGE typing assigned all laboratory strains into cluster Q, which shared 49% similarity with clusters A and B, and 35% similarity with cluster C. Automated ribotyping placed laboratory strains within ribogroups different from those of bovine isolates. These methods have 70% concordance and permitted identification of the prototypic vaccine background from those of clinical isolates. This information is required before conducting field trials with the vaccine.

Genotypic analysis of Staphylococcus aureus from milk of dairy cows with mastitis in Argentina.

Staphylococcus aureus is the most prevalent pathogen causing mastitis of dairy ruminants. This study was developed to ascertain the genotypes and genealogical relationship among strains isolated from milk of bovines with mastitis in Argentina. Molecular epidemiological analysis of S. aureus was performed on 112 isolates from 21 districts. Clonality was assessed by SmaI pulsed-field gel electrophoresis (PFGE) typing, automated EcoRI ribotyping and restriction enzyme analysis of plasmid (REAP) DNA profiles. A total of 22 band patterns distributed in four clusters were found by SmaI PFGE analysis. The similarity of clusters 2, 3 and 4 with cluster 1 was 0.73, 0.69 and 0.33, respectively, and 101 of 112 isolates belonged in cluster 1. PFGE band patterns from 42 isolates within cluster I were indistinguishable from each other (type A). The second largest group of isolates with indistinguishable PFGE band patterns was subtype A11, which was composed of 19 isolates. Automated ribotyping assigned the 112 isolates into 13 ribotypes. Among these, the most prevalent ribotypes I and VI were composed of 49 and 35 isolates respectively. Although there was certain correspondence between PFGE genotypes and ribotypes, further discrimination was achieved by combining both methods. REAP DNA profile analysis was useful to provide even further discrimination between isolates with identical PFGE genotype and ribotype. The most prevalent S. aureus strains A/I and A11/VI were widely distributed in the country and were not restricted to individual nearby locations. Prevalence of these two strains varied consecutively within a period of 8 years. Whether the shift in type prevalence was due to selection of a phenotypic trait remains undisclosed.