Zuzana Cermakova

Laboratory diagnosis of leptospirosis.

Percentage of serological positivity examined in 4205 blood sera by serological method microscopic agglutination test (MAT) on the hinterland territory of our laboratory (East Bohemia; 1999–2003) was 0.38–4.7 %. By the PCR method for detection of DNA of pathogenic leptospires (L. interrogans, L. borgpetersenii andL. kirschneri) from 57 samples of different biological materials from patients with fever of unknown etiology positive results were obtained in 4 specimens (7 %; 3 samples of urine and 1 sample of blood). This method was shown to distinguish between pathogenic and nonpathogenic strains and can detect 2.5–10 cells per mL of biological material. As an important presumption of successful detection of pathogenic leptospires a correct collecting of blood, urine samples or liquor is required before starting antibody therapy. The PCR method possesses a clear advantage over other methods, such as MAT, which relies on the detection of antibodies the presence of which cannot be detected until days after infection.

Real-time PCR method for the detection of the gene encoding surface lipoprotein LipL32 of pathogenic Leptospira: use in the laboratory diagnosis of the acute form of leptospirosis.

Abstract Background: The aims of this work were to replace the obsolete PCR method for the laboratory diagnosis of the acute form of leptospirosis using the G1, G2 and B64 I, B64 II primers, and to improve the PCR detection time. Methods: We introduced a real-time PCR method for the detection of the gene encoding the surface lipoprotein LipL32 of pathogenic Leptospira into our laboratory diagnosis of the acute form of leptospirosis. The positive and negative analytical specificities of the real-time PCR method were both equal to 100%; the detection limit was determined to be 1–5 genome copies/1 ml of liquid biological material. The method was further validated on 230 laboratory strains of leptospires. Results: All laboratory strains of pathogenic Leptospira were evaluated as LipL32-positive and all non-pathogenic strains as LipL32-negative. In addition, 455 biological materials (253 plasma, 121 urine, 72 cerebrospinal fluid (CSF), 7 bronchoalveolar lavage, and 2 sputum) from 295 patients with suspected leptospirosis were examined. From this set of patients, 9 were evaluated to be LipL32-positive, from 15 positive biological materials (10 urine, 4 blood plasma, and 1 CSF). Conclusions: This real-time PCR method for the detection of the gene encoding the surface lipoprotein LipL32 is a reliable, sensitive, and rapid method for the detection of the acute form of leptospirosis.

Polymerase chain reaction for detection ofToxoplasma gondii in human biological samples

Using the polymerase chain reaction (PCR),Toxoplasma gondii from gene TGR 1E with primers TGR1E-1, TGR1E-2 (standard PCR), and from B1 gene with primers TM1, TM2, TM3 (hemi-nested PCR) was detected in biological samples from 347 individuals (441 biological materials). Of the total of 441 biological materials,T. gondii DNA was detected in 5.2 %; it was positive in the following samples: blood (n=6), blood from newborns (2), biopsies (2) and samples of progenitor cells (2) (from candidates for bone marrow transplantation). DNA ofT. gondii was also revealed in 11 samples (8.3 %) of 120 cases of pregnant women during prenatal examinations. A positive result in the blood was also found in two cases of newborn babies from mothers who were infected in later pregnancy. The positive PCR examination was confirmed by serological methods (ELISA and complement fixation test). Agreement of PCR results and the detection of antibodies against toxoplasma was found in 83.3 %. Rapid PCR examination for the confirmation of acute parasitemiaT. gondii is particularly important for the patients in whom the infection may cause serious consequences (e.g., for fetus in pregnant women or for patients suffering from imunosuppression).

Leptospirosis: possibilities of early laboratory and clinical diagnosis

This retrospective study aims to identify and describe the problems associated with the laboratory and clinical diagnosis of leptospirosis. A total of 4,813 patients with suspected leptospirosis from an area of the Czech Republic, with a total population of 1.15 million, were examined during the period 2002–2010. Our study included only 855 patients: 545 men (mean age 41.03 ± 19.24) and 310 women (mean age 41.47 ± 20.3) who were examined using microscopic agglutination test (MAT) and a polymerase chain reaction (PCR). All patients and their physicians filled in questionnaires, which included anamnestic data, clinical symptoms and the results of laboratory tests. Out of total suspected, 89 patients (1.85%), tested positive for leptospirosis, of which 50 have been examined only serologically by MAT. Of 855 patients in our study undergoing both PCR and MAT tests, 39 have tested positive for leptospirosis. The most frequent symptom in patients with leptospirosis included fever (91.6%) and headache (69.4%). The correct laboratory diagnosis of leptospirosis depends on biological material being tested before the start of antibiotic treatment, since leptospires are extremely sensitive to antibiotics. Consequently, the PCR results alone may produce a false negative result after 24 hours following treatment with antibiotics.

Our experience using real-time PCR for the detection of the gene that encodes the superficial lipoprotein LipL32 of the pathogenic leptospires to confirm the acute form of human leptospirosis

AIMSnTo examine biological materials (blood, urine, cerebrospinal fluid) of patients with suspected leptospirosis using real-time PCR for detecting the gene that codes the superficial LipL32 lipoprotein, and to evaluate the contribution of the real-time PCR method for the laboratory diagnosis of the acute form of leptospirosis.nnnMATERIAL AND METHODSnDuring the monitored period of April 2010 - December 2011, a total of 340 biological materials samples were examined (177x blood plasma, 88x urine, 68x, cerebrospinal fluid, 6x bronchoalveolar lavage and 1x sputum) from 216 patients with suspected leptospirosis using real-time PCR LipL32 gene detection.nnnRESULTSnFrom the mentioned 216 patients suspected of leptospirosis, 8 patients were evaluated as being PCR LipL32 positive, from which 14 positive biological materials originated (9 x urine, 4x blood and 1x liquor).nnnCONCLUSIONnAs demonstrated in the study, the real-time PCR method for detecting the gene for the superficial lipoprotein LipL32 is an appropriate, quick and reliable method for the diagnosis of the acute form of leptospirosis.

Leptospirosis: a neglected zoonosis of global distribution

This review details the basic characteristics about a neglected zoonosis of worldwide distribution – leptospirosis. Prevalence of infection caused by pathogenic leptospires ranges from 1 to 100 per 100u200a000 inhabitants. Leptospirosis can manifest itself as a mild flu-like illness associated with sudden fever, headache and myalgia. In 90% of cases, anicteric forms of the disease develop and in the remaining 10% of cases severe icteric course of infection is seen. More than 500u200a000 cases of severe leptospirosis are described annually with mortality from 5 to 15%. This review will primarily focus on the pathogenesis, the clinical manifestations, the laboratory diagnosis and treatment of this infectious disease.