Culture-free genome-wide locus sequence typing (GLST) provides new perspectives on Trypanosoma cruzi dispersal and infection complexity.

Analysis of genetic polymorphism is a powerful tool for epidemiological surveillance and research. Powerful inference from pathogen genetic variation, however, is often restrained by limited access to representative target DNA, especially in the study of obligate parasitic species for which ex vivo culture is resource-intensive or bias-prone. Modern sequence capture methods enable pathogen genetic variation to be analyzed directly from host/vector material but are often too complex and expensive for resource-poor settings where infectious diseases prevail. This study proposes a simple, cost-effective 'genome-wide locus sequence typing' (GLST) tool based on massive parallel amplification of information hotspots throughout the target pathogen genome. The multiplexed polymerase chain reaction amplifies hundreds of different, user-defined genetic targets in a single reaction tube, and subsequent agarose gel-based clean-up and barcoding completes library preparation at under 4 USD per sample. Our study generates a flexible GLST primer panel design workflow for Trypanosoma cruzi, the parasitic agent of Chagas disease. We successfully apply our 203-target GLST panel to direct, culture-free metagenomic extracts from triatomine vectors containing a minimum of 3.69 pg/µl T. cruzi DNA and further elaborate on method performance by sequencing GLST libraries from T. cruzi reference clones representing discrete typing units (DTUs) TcI, TcIII, TcIV, TcV and TcVI. The 780 SNP sites we identify in the sample set repeatably distinguish parasites infecting sympatric vectors and detect correlations between genetic and geographic distances at regional (< 150 km) as well as continental scales. The markers also clearly separate TcI, TcIII, TcIV and TcV + TcVI and appear to distinguish multiclonal infections within TcI. We discuss the advantages, limitations and prospects of our method across a spectrum of epidemiological research.

Incidence, mortality, and prevalence of end-stage chronic renal disease in the Bajo Lempa region of El Salvador: A ten-year community registry. / Incidencia, mortalidad y prevalencia de enfermedad renal crónica terminal en la región del Bajo Lempa, El Salvador: 10 años de registro comunitario.

The Bajo Lempa is an impoverished rural coastal region of El Salvador affected by the chronic kidney disease (CKD) epidemic known as Mesoamerican nephropathy. The local community organisation Fondo Social de Emergencia para la Salud (FSES) (Emergency social fund for health) is helping to fight the epidemic in 42 communities of the region (19,223 inhabitants; average age 26.7 years; 48.5% male; 40.2% <18 years). OBJECTIVES: To report annual rates of end-stage renal disease (ESRD) incidence and patient mortality in these communities during a 10-year period (2004-2013), and the prevalence of patients receiving renal replacement therapy (RRT) as of 31 December 2013. METHODS: The FSES recorded new ESRD cases, basic patient history, form of RRT if received and patient deaths. RESULTS: We registered 271 new ESRD cases (annual average 27.1; 89% male; average age 55.6 years, four <18 years). Average annual ESRD incidence rate: 1409.8 per million population (pmp). Two-thirds did not report diabetes or hypertension. 94 patients (34.7%) received RRT: 58 in the Ministry of health, 26 in private services, 9 in social security and 1 in the military health system. 246 patients died (annual average 24.6 deaths; 89.4% male; average age 56.1 years; 92.3% at home). Average annual mortality rate: 128/100,000 population. Prevalence of patients receiving RRT in 2013: 1300.5 pmp (N=25; 84% male; average age 51 years). CONCLUSIONS: This region has a high incidence of ESRD. Few receive RRT. Patient mortality is high even with RRT. Most patients are male (9:1). Social determinants influence the high mortality.

EURADOS intercomparison on emergency radiobioassay.

Nine laboratories participated in an intercomparison exercise organised by the European Radiation Dosimetry Group (EURADOS) for emergency radiobioassay involving four high-risk radionuclides ((239)Pu, (241)Am, (90)Sr and (226)Ra). Diverse methods of analysis were used by the participating laboratories for the in vitro determination of each of the four radionuclides in urine samples. Almost all the methods used are sensitive enough to meet the requirements for emergency radiobioassay derived for this project in reference to the Clinical Decision Guide introduced by the NCRP. Results from most of the methods meet the requirements of ISO 28218 on accuracy in terms of relative bias and relative precision. However, some technical gaps have been identified. For example, some laboratories do not have the ability to assay samples containing (226)Ra, and sample turnaround time would be expected to be much shorter than that reported by many laboratories, as timely results for internal contamination and early decisions on medical intervention are highly desired. Participating laboratories are expected to learn from each other on the methods used to improve the interoperability among these laboratories.

[Assessment of two DNA extraction methods to amplify the pneumolysin gene (PLY) from blood culture samples of Streptococcus pneumoniae]. / Evaluación pre-analítica de dos métodos de extracción de ADN para la amplificación del gen de la pneumolisina (PLY) de Streptococcus pneumoniae, en muestras de hemocultivo.

BACKGROUND: Streptococcus pneumoniae is a common etiologic agent of invasive respiratory infections among children under 5 years of age and older adults. Isolation rates of S. pneumoniae by traditional culture techniques are low. AIM: To study the sensitivity and specificity of two different DNA extraction methods to amplify the ply gene, applied to three different types of blood culture broths, experimentally inoculated with S. pneumoniae. MATERIAL AND METHODS: DNA was extracted from the cultures using an organic method or a technique that consists in dilution, washing with NaOH and concentration of the sample. This was followed by PCR amplification of a 355 pb fragment of the pneumolysin gene (ply). RESULTS: The organic DNA extraction method inhibited the PCR reaction at all concentrations studied (0.6 to 10(6) colony forming units/mL). Using the NaOH extraction, ply gene amplification was positive in all three blood culture broths, but only at concentrations of 10(3) colony forming units/mL, or higher. Using the same DNA extraction method, PCR was negative when the broths were inoculated with seven other related bacterial species, which results in a 100% specificity. CONCLUSIONS: Detection of S. pneumoniae by amplification of ply gene from blood cultures using the protocol of NaOH for DNA extraction is specific and provides results in a short lapse. However, the diagnostic sensitivity is not optimal, which limits its clinical use.