Fig. 1 | Scientific Reports

Fig. 1

From: In-house isolation protocol from human serum samples demonstrates the circulating of a broad diversity of Leptospira serogroups in Costa Rica

Fig. 1

Stepwise protocol for the isolation of Leptospira from serum human samples. (1) Patient with febrile symptoms compatible with leptospirosis and with 1–6 days from the onset of symptoms. (2). Draw blood into collection tubes without additives. (3) Centrifugation of blood sample in a bench centrifuge at 3500 rpm for 10 min to obtain serum. (4) Storage of serum samples at 4–8 °C until further analysis. (5) Extraction of genomic DNA from 200 µl of serum samples. (6) Real time PCR targeting the Leptospira-secY gene (a Ct of 40 was considered positive when the Tm value of the amplicon from 2 or more replicates did not vary by more than 0.5 °C). (7) Five drops of each of the qPCR-positive serum samples were inoculated in 6 ml of Ellinghausen-McCullough-Johnson-Harris (EMJH) medium supplemented with albumin-fatty-acid-supplement (AFAS) and in EMJH medium supplemented with sulfamethoxazole-trimethoprim-amphotericin-fosfomycin-5-fluorouracil (STAFF) and AFAS. (8) The culture tubes were incubated at 30 °C for 6 months until turbidity was observed. (9) Once a tube becomes turbid, the culture was examined by dark field microscopy to determine the presence of leptospires. (10) Cultures confirmed positive by dark-field microscopy were further characterized using microagglutination test (MAT) and whole-genome sequencing (WGS).

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