Detection and genotyping of oocysts of Cryptosporidium parvum by real-time PCR and melting curve analysis

Several real-time PCR procedures for the detection and genotyping of oocysts of Cryptosporidium parvum were evaluated. A 40-cycle amplification of a 157-bp fragment from the C. parvum beta-tubulin gene detected individual oocysts which were introduced into the reaction mixture by micromanipulation. SYBR Green I melting curve analysis was used to confirm the specificity of the method when DNA extracted from fecal samples spiked with oocysts was analyzed. Because C. parvum isolates infecting humans comprise two distinct genotypes, designated type 1 and type 2, real-time PCR methods for discriminating C. parvum genotypes were developed. The first method used the same beta-tubulin amplification primers and two fluorescently labeled antisense oligonucleotide probes spanning a 49-bp polymorphic sequence diagnostic for C. parvum type 1 and type 2. The second genotyping method used SYBR Green I fluorescence and targeted a polymorphic coding region within the GP900/poly(T) gene. Both methods discriminated between type 1 and type 2 C. parvum on the basis of melting curve analysis. To our knowledge, this is the first report describing the application of melting curve analysis for genotyping of C. parvum oocysts.     

Is the Parkland formula still the best method for determining the fluid resuscitation volume in adults for the first 24 hours after injury? — A retrospective analysis of burn patients in Germany

BackgroundR Rapid fluid resuscitation is a crucial therapy during the treatment of patients with extensive burns. In 1968, the Parkland Formula was introduced for the calculation of the estimated volume of the resuscitation fluid. Since then, different methods for the calculation of fluid resuscitation volume have been developed. We aimed to evaluate if the Parkland formula is still the most effective method for fluid resuscitation volume calculation in burn patients.MethodsIn the period between January 2015 and January 2019, data from 569 patients over 16 years old with burns of more than 20% total body surface area (TBSA) and at least 15% TBSA full thickness burns were entered in the German burn registry. The patients were divided into 5 groups (0, +1, ?1, +2, ?2) according to the volume of the resuscitation fluid they received. Group 0 patients received the amount of fluid calculated according to the Parkland formula (n = 83). The 4 other groups received reduced (-1, -2) or increased (+1, +2) fluid volumes in comparison to the value obtained by the Parkland formula.ResultsPatients in Group 0 presented a significantly lower mortality in the first week (4.5%) compared to groups –2 (16.7%) and group +2 (19.5%) (p = 0.021). Furthermore, the mean number of operations in group +2 (5.81) was higher than in group ?2 (3.81). Surviving patients from group +2 presented a longer hospital stay (68.1 days) compared to the other groups. Additionally, the logistic regression analysis showed a higher survival of patients in groups ?2 and ?1 (regression coefficients ?0.11 and ?0.086; Odds Ratio 0.896 and 0.918; 95% Confidence Interval (CI) 0,411–1.951 and 0.42–2.004).ConclusionIn this retrospective study, register based analysis a restrictive fluid regime was associated with a higher survival compared to the liberal Parkland guided fluid regime.     

Survivin and aven: two distinct antiapoptotic signals in acute leukemias.

BACKGROUND: Antiapoptotic signals are important in the development, progression and prognosis of malignant tumors. The aim of this study was to determine the two distinct antiapoptotic signals-survivin and aven-in acute leukemias and compare them with clinical and hematological findings and response to therapy. Real-time quantitative PCR was used and survivin and aven were detected at the messenger (m)RNA level. PATIENTS AND METHODS: Sixty-five patients with acute leukemia [37 with acute myeloblastic leukemia (AML) and 28 with acute lymphoblastic leukemia (ALL)] were used as the study group and 10 healthy subjects were used as the control group. RESULTS: Survivin was between 0.0 and 0.829 copy number/cell (median 0.0721, mean 0.5424301909 +/- 0.139799488589) and aven was between 0.0 and 0.853 copy number/cell (median 0.0124, mean 0.070335542 +/- 0.1524685709). We found an important association between survivin and aven (P = 0.000). Both survivin and aven were higher in the study group than in the controls (P = 0.001 and 0.035, respectively). When we compared survivin and aven with other clinical and hematological parameters, there was an important association between survivin and extramedullary involvement (P = 0.033), survivin and alkaline phosphatase (P = 0.06), white blood cell (WBC) count and lactate dehydrogenase (LDH) (P = 0.000), WBC count and uric acid (P = 0.074), hemoglobin level and LDH (P = 0.072), LDH and uric acid (P = 0.057), CD7 expression and survivin (P = 0.097), and CD34 expression and aven (P = 0.058). Response to therapy was evaluated according to the survivin and aven levels. Survivin level was lower in refractory patients as compared with complete responders (P = 0.085). Aven level was higher in patients with relapse as compared with non-relapse patients (P = 0.04). There was no important association between survivin or aven and performance status, lymphadenopathy or organomegaly. CONCLUSIONS: Both survivin and aven are important antiapoptotic signals in acute leukemias, and the association between extramedullary involvement, CD7 expression and CD34 expression, which are important poor prognostic indicators in acute leukemias, suggests that survivin and/or aven may be novel prognostic indicators in acute leukemias. Further studies with a higher number of patients will be more informative.     

The evaluation of Chronic Progressive External Ophthalmoplegia with computerized tomography

Introduction: Chronic progressiveexternal ophthalmoplegia is characterisedby limitation of ocular motility in alldirections of gaze and ptosis.Innervational or myogenic factors wereclaimed to be responsible for thismotility disorder. The aim of thisstudy was toinvestigate the extraocular muscles inCPEO with computerized tomography in an attemptto distinguish extraocular musclemorphology caused by this disorderfrom that occurring in normal individuals.Methods: Eighteen orbits from 9patients diagnosed with CPEOwere included in the study. Axialand coronal scans were obtained for CTevaluation of extraocular muscles and thedimensions of extraocular muscles were measured.The control group consisted of40 orbits belonging to 20 individuals and,the results were compared with a student'st test.Results: The thickness (the verticaldimension of vertical recti and thehorizontal dimension of horizontal recti)of all rectus muscles wassignificantly decreased in comparison with the controlgroup, whereas the width (the horizontal dimensionof vertical recti and the verticaldimension of horizontal recti) was similar inboth the diseased and normal orbits.In all the rectus musclesof the diseased orbits,the normal fusiform shape was lost and the muscles appeared asthin bands.Discussion: The differentiation ofCPEO from other myogenic and neurogenicdisorders may present difficulty,and a cluster of criteria are required fora final diagnosis. CT has provento be a valuable tool in assessing extraocularmuscles [1, 2]. In this study, an extremeatrophy of all rectus muscles wasdemonstrated by means of CT. This diagnostic method mayconsequently contribute to a properdiagnosis of CPEO.     

Is there any effect of bolus and/or infusion 5-fluorouracil treatment on microalbuminuria in immediate or long term?

5-Fluorouracil is a widely used cytotoxic chemotherapeutic agent in the treatment settings particularly in patients with gastrointestinal cancer. Various studies on the cardiac adverse effects of 5-fluorouracil, reported the likelihood of altered myocardial contractility and vascular endothelial damage caused by this agent. However, the mechanism underlying 5-fluorouracil-related cardiotoxicity is not clear. In certain experimental studies, thrombotic processes occurring in microvascular field were supposed to play a role in this condition. In the light of this knowledge, the administration of 5-fluorouracil may be considered to cause renal vascular endothelial damage that may result in the altered endothelial permeability. As a result of endothelial dysfunction, increased urinary albumin excretion may be in question and no study investigating this potential direct relationship has been available in medical literature. Based on this evidence, the hypothesis of that 5-fluorouracil might cause renal vascular dysfunction and microalbuminuria, was discussed in this article along with the basic knowledge.