钩端螺旋体赖株豚鼠感染模型的建立和免疫组化法对钩体抗原的检测

目的　建立钩端螺旋体赖型赖株感染豚鼠的动物模型 ,并用免疫组化法检测钩端螺旋体抗原在组织中的分布。方法　不同剂量钩端螺旋体赖株通过腹腔内注射感染豚鼠 ,观察豚鼠发病情况 ,及时解剖死亡豚鼠 ,HE染色观察肝、肾、肺等器官病变 ,EnVision二步法染色检测肝、肾、肺组织中钩体抗原。结果　感染后豚鼠发病 ,死亡与感染的钩端螺旋体剂量明显相关 ,大体标本及HE染色光镜下均出现典型钩体病病变。免疫组化显示在肝、肾、肺组织中均存在钩体抗原。结论　通过钩端螺旋体赖株腹腔注射的方法成功地建立了豚鼠动物模型。应用EnVision二步法染色 ,可有效显示组织中的钩体抗原。为进一步研究钩端螺旋体发病机制奠定了基础。     

Disseminated intravascular coagulation complicated by peripheral gangrene in a rhesus macaque (Macaca mulatta) experimentally infected with Plasmodium coatneyi

We report the first case of disseminated intravascular coagulation (DIC) complicated by peripheral gangrene induced by Plasmodium coatneyi in rhesus monkeys. Ten days after experimental challenge, numerous petechiae were noted over the trunk and extremities, with polychromasia, severe anemia, thrombocytopenia, and moderate parasitemia. These changes were accompanied by elevated serum activity of blood urea nitrogen, creatinine, transaminases, and creatinine phosphokinase. The animal received intravenous fluid support, artemether, and blood transfusion. Three days after treatment, the platelet counts returned to normal, and parasitemia was abated. However, several areas of skin discoloration with gangrenous tissue in the hands and the tail were observed. Coagulation profile showed elevated D-dimers and elevated levels of fibrinogen/fibrin degradation products with low levels of protein S functional activity. DIC with peripheral gangrene is very rare in Plasmodium-infected individuals. Our results indicate that the experimental model of P. coatneyi infection of rhesus monkeys is important for studies of malarial anemia and coagulopathy.     

Clinical and histologic features of skin lesions in a cynomolgus monkey experimentally infected with mycobacterium ulcerans (Buruli ulcer) by intradermal inoculation

Buruli ulcer, caused by Mycobacterium ulcerans, is a destructive infection that most commonly affects the skin. Animal models for Buruli ulcer include guinea pigs, rats, mice, and armadillos, but each is limited in replicating the spectrum of human disease. Here, a cynomolgus monkey was infected with two concentrations of M. ulcerans (1.0 and 2.2 x 10(8)) by intradermal inoculation, 3 months apart. All injection sites developed papules that progressed to ulcers with undermined borders within 2-4 weeks. The rate of progression and size of the ulcers were proportional to the numbers of organisms inoculated. Biopsies from ulcer edges showed ulceration, robust inflammatory cell infiltrates, granulomatous-like responses, mild edema, and extracellular acid-fast bacilli. The ulcers healed spontaneously between Weeks 8 and 12, with no signs of systemic infection. This report, the first to describe a non-human primate experimentally infected with M. ulcerans, suggests that cynomolgus monkeys are modestly susceptible and develop some of the clinical and histologic features of Buruli ulcer.     

Platelet-associated autoantibodies as detected by a solid-phase modified antigen capture ELISA test (MACE) are a useful prognostic factor in idiopathic thrombocytopenic purpura

There were 50 consecutive idiopathic thrombocytopenic purpura (ITP) adult patients (platelet count < 100 x 10(9)/L) grouped according to positivity or negativity of a solid-phase modified antigen capture enzyme-linked immunosorbent assay (ELISA) test (MACE) against glycoprotein IIb/IIIa (GPIIb/IIIa), Ib/IX, and IIa/IIIa. Observation started on the day of MACE assay and lasted at least 6 months. Clinical worsening was defined as the need for starting or modifying therapy because of thrombocytopenia lower than 20 x 10(9)/L or patient admission due to bleeding symptoms. MACE-positive patients had a higher probability of clinical worsening than MACE-negatives (P <.004). The proportion of patients worsening was 18 (72%) of 25 among MACE-positives and 8 (32%) of 25 among MACE-negatives. The median time to clinical worsening was 2.1 months for MACE-positive patients and 27.7 months for MACE-negatives. The assay of specific platelet autoantibodies may be a useful prognostic tool for the clinical course of ITP.     

Clinical laboratory, virologic, and pathologic changes in hamsters experimentally infected with Pirital virus (Arenaviridae): a rodent model of Lassa fever

The clinical laboratory, virologic, and pathologic changes occurring in hamsters after infection with Pirital virus (Arenaviridae) are described. Pirital virus infection in the hamsters was characterized by high titered viremia, leukocytosis, coagulopathy, pulmonary hemorrhage and edema, hepatocellular and splenic necrosis, and marked elevation of serum transaminase levels. All of the animals died within 9 days. The clinical and histopathological findings in the Pirital virus-infected hamsters were very similar to those reported in severe human cases of Lassa fever, suggesting that this new animal model could serve as a low-cost and relatively safe alternative for studying the pathogenesis and therapy of Lassa fever.     

Thromboembolic risk evaluation in patients with atrial fibrillation using a modified CHADS(2) scoring system

Thromboembolic Risk Stratification in AF. Background : Antithrombotic recommendations for relatively low risk patients with atrial fibrillation (AF) are not well established. Some patients with CHADS₂ score = 0 have a CHA₂DS₂–VASc score of 2, which indicated warfarin therapy in the latter system. We evaluated the thromboembolic risk in AF patients with a CHADS₂ score of 0 or 1. Methods: A total of 695 patients with AF that were followed for ≥ 12 months (median 65.6 months, range 12–138 months), were analyzed retrospectively. The modified CHADS₂ score (MCS) was applied as follows. Each CHADS₂ score group was divided into 2 groups, A and B (i.e., MCS 0A vs 0B, and MCS 1A vs 1B) according to the number of nonmajor risk factors (female gender, chronic kidney disease, coronary artery disease, age 65–74 years). Group A had 0 or 1, and group B had 2 or more nonmajor risk factors. Results: In patients with CHADS₂ score = 1, there were 13 thromboembolic events (0.65%/year) in 343 MCS 1A patients, and 12 thromboembolic events (1.90%/year) in 108 MCS 1B patients. Thromboembolic risk was significantly higher in the MCS 1B compared to the MCS 1A patients (P = 0.006). In 244 patients with CHADS₂ score = 0, the thromboembolic risk of MCS 0B was similar to that of MCS 0A (P = 0.095), and 26 patients had a CHA₂DS₂–VASc score of 2. Conclusion: Patients with MCS 1B had a higher thromboembolic risk than patients with MCS 1A. Antithrombotic strategies for patients with a CHA₂DS₂–VASc score of 2 but a CHADS₂ score of 0 need further investigation. J Cardiovasc Electrophysiol, Vol. 23, pp. 155‐162, February 2012)     

MRI detection of glycogen in vivo by using chemical exchange saturation transfer imaging (glycoCEST)

Detection of glycogen in vivo would have utility in the study of normal physiology and many disorders. Presently, the only magnetic resonance (MR) method available to study glycogen metabolism in vivo is (13)C MR spectroscopy, but this technology is not routinely available on standard clinical scanners. Here, we show that glycogen can be detected indirectly through the water signal by using selective radio frequency (RF) saturation of the hydroxyl protons in the 0.5- to 1.5-ppm frequency range downfield from water. The resulting saturated spins are rapidly transferred to water protons via chemical exchange, leading to partial saturation of the water signal, a process now known as chemical exchange saturation transfer. This effect is demonstrated in glycogen phantoms at magnetic field strengths of 4.7 and 9.4 T, showing improved detection at higher field in adherence with MR exchange theory. Difference images obtained during RF irradiation at 1.0 ppm upfield and downfield of the water signal showed that glycogen metabolism could be followed in isolated, perfused mouse livers at 4.7 T before and after administration of glucagon. Glycogen breakdown was confirmed by measuring effluent glucose and, in separate experiments, by (13)C NMR spectroscopy. This approach opens the way to image the distribution of tissue glycogen in vivo and to monitor its metabolism rapidly and noninvasively with MRI.     

Demonstration by immunocytochemical staining of a somatostatin-28-(1-14)-like peptide in planarians (Plathyhelminthes Turbellaria Tricladida)

A specific polyclonal antiserum directed against the somatostatin-28(1-14) of vertebrates was applied to sections of the planarians Dugesia lugubris and Dendrocoelum lacteum. This made it possible to reveal nerve cells and processes specifically both in cerebral ganglia and in ventral nerve cords. The phylogenetic importance of this demonstration is pointed out.     

Early detection of orthostatic hypotension by quantitative sudomotor axon reflex test (QSART) in type 2 diabetic patients

OBJECTIVE: Orthostatic hypotension is caused by autonomic nerve dysfunction, mainly by severe sympathetic nerve dysfunction in diabetic patients. Diabetes affects the peripheral nerves in a length-dependent manner. Quantitative sudomotor axon reflex test (QSART) is one of the sensitive tests for detecting sympathetic nerve function. We examined the relation between orthostatic hypotension and QSART at the foot and hand in type 2 diabetic patients. METHODS: Thirty-eight type 2 diabetic patients (age, 48.9 +/- 11.9 years; duration of diabetes, 13.4 +/- 8.6 years) and 13 age-matched non-diabetic controls were evaluated. All subjects aged under 65 years old were recruited. All subjects underwent Schellong tests and quantitative sudomotor axon reflex tests (QSART) at the back of the hand and dorsum of the foot. RESULTS: The sweating volume at the foot dorsum, but not the back of the hand, during the first 10 minutes of QSART was significantly related to the orthostatic hypotension on the Schellong test. In patients with normal, borderline and abnormal blood pressure response to standing, 6 out of 17 (35.3%), 9 out of 12 (75.0%) and 9 out of 9 (100%) had decreased sweating volume of the foot dorsum, respectively. CONCLUSIONS: Our results suggest that orthostatic hypotension may be detected early by QSART at the dorsum of the foot in type 2 diabetic patients.     

Visceral leishmaniasis caused by Leishmania (Viannia) braziliensis in a patient infected with human immunodeficiency virus

The current article reports the case of a 19-month-old-girl, from the state of Minas Gerais, Brazil, with visceral leishmaniasis, by Leishmania (Viannia) braziliensis, and Human Immunodeficiency Virus (HIV) co-infection. The child's mother and father, aged 22 and 27 years old, respectively, were both HIV positive. The child was admitted to the General Pediatric Center, in Belo Horizonte, presenting high fever, fatigue, weight loss and enlargement of liver and spleen. Indirect immunofluorescent test revealed a titer of 1:320 for Leishmania. Such result was confirmed by the presence of amastigotes in bone marrow aspirate samples and culture of promastigote forms. Parasites were identified as being Leishmania (Viannia) braziliensis through PCR, using a L. braziliensis complex primer and a generic primer, followed by hibridization. Specific leishmaniasis therapy (Glucantime register mark or target antimonial) was intravenously administered.     

Cell lineage specific involvement in acute promyelocytic leukaemia (APL) using a combination of May-Grünwald-Giemsa staining and fluorescence in situ hybridization techniques for the detection of the translocation t(15;17)(q22;q12)

Acute promyelocytic leukaemia (APL) is strongly associated with the translocation t(15;17) which therefore provides a reliable marker to assess the potential involvement of different cell lineages. Six cases with morphologically, cytogenetically and molecularly proven APL were analysed at diagnosis or relapse by combining fluorescence in situ hybridization (FISH) with standard May-Grünwald-Giemsa (MGG) staining at the single cell level on bone marrow and blood smears. With the FICTION technique, combining immunophenotyping with FISH, haemopoietic precursor cells were identified using monoclonal antibodies against CD34, B- and T-lymphocytes could be identified with CD19 and CD3. In addition, HLA-DR-positive cells were studied for the presence of t(15;17).
Morphologically identified myeloblasts were relocated on the smear after FISH and were found to be PML/RARA positive in 91%, abnormal promyelocytes in 97%. In contrast, a positive signal was obtained in only 18% of PMN. Erythroblasts, lymphocytes and plasma cells did not show a PML/RARA rearrangement. Accordingly, all cells expressing CD3 or CD19 were PML/RARA negative. CD34 positive precursor cells identified by FICTION were PML/RARA positive in 97%. HLA-DR-positive cells contained a PML/RARA rearrangement in 24% of cells in one case and were negative in the two other cases investigated. These data indicate that APL appears to originate from a level of haemopoietic precursor cells but is restricted to the myeloid lineage. The low proportion of PML/RARA-positive PMN points to the impairment of blast cell differentiation beyond the promyelocyte stage but also emphasizes the existence of normal residual haemopoietic stem cells from which PML/RARA-negative PMN must be derived.     

Detection of giardia antigen in stool samples by a semi-quantitative enzyme immunoassay (EIA) test

A semi-quantitative enzyme immunoassay (EIA) test for the detection of Giardia intestinalis antigen in faeces was developed. In order to avoid unspecific reactions due to anticalf serum activity, IgG fractions of anti-giardia rabbit and sheep sera were purified from antibovine antibodies by immunoadsorption. Faecal specimens tested in the assay were mixed with normal horse serum to avoid unspecific and proteolytic effects of stool components. Out of a range of bacterial and parasitic antigens tested, only high concentrations of Ascaris suum egg antigen and Shigella sonnei gave slight unspecific reactions. During an outbreak of waterborne giardia infection faecal samples from 49 individuals were tested. Giardia cysts were found by microscopy in 22 individuals and specimens from 24 persons were positive by EIA. The estimated amount of antigen found in the positive samples ranged from 50 to 5,000 ng giardia protein/ml. After filtration through a 0.45 microns filter antigenic activity was found in the filtrate of 10 samples. In 3/6 samples with no cysts detected by microscopy, mainly soluble giardia antigen was found.     

Value of post-vascular phase (Kupffer imaging) by contrast-enhanced ultrasonography using Sonazoid in the detection of hepatocellular carcinoma

Background

We evaluated the sensitivity and specificity of post-vascular phase (Kupffer imaging) by contrast-enhanced ultrasonography (CEUS) using perflubutane microbubbles (Sonazoid) in comparison with conventional B-mode ultrasonography (US) for the detection of hepatocellular carcinoma (HCC) nodules.

Methods

A total of 100 treatment-na?ve HCC patients admitted at our hospital between December 2007 and June 2009 were consecutively enrolled. The sensitivity and specificity of conventional and contrast-enhanced US were evaluated on a liver segment basis using dynamic CT as a reference standard. Movie files of conventional and enhanced US were stored separately for each segment (e.g., lateral, medial, anterior, and posterior) and reviewed randomly by two blinded readers.

Results

A total of 138 HCC nodules (mean diameter 20.3?mm) were detected in 123 of 400 segments. Detection sensitivity of B-mode US was 0.837 for reader A and 0.846 for reader B, and that of CEUS was 0.732 for reader A and 0.831 for reader B. Specificity of B-mode US was 0.902 for reader A and 0.949 for reader B, and that of CEUS was 0.986 for reader A and 0.978 for reader B. CEUS false positives were mainly due to misidentification of hepatic cysts. A significant proportion of false-negative nodules are hyperechoic in B-mode US, likely because echogenicity hampers visualization of the defect in Kupffer imaging.

Conclusions

Kupffer imaging by CEUS with Sonazoid showed very high specificity but rather mediocre sensitivity for HCC detection. CEUS is highly suitable for confirmatory diagnosis of HCC; however, caution should be exercised in reaching a diagnosis based only on CEUS.     

Recurrent PIG-A mutation (IVS5+1G→A) in a paediatric case of paroxysmal nocturnal haemoglobinuria: detection by the protein truncation test

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haemopoietic stem cell disorder caused by the absence of glycosyl phosphatidylinositol (GPI)-anchored surface proteins due to a deficient biosynthesis of GPI-anchor. The disease occurs predominantly in adults, and very few cases have been described in children and adolescents. Recent analyses have shown that null mutations in the X-linked PIG-A (phosphatidylinositol glycan-class A) gene are responsible for GPI-anchor deficiency in most PNH adult patients analysed. We report a young male from southern France who was diagnosed with PNH at 12 years of age during follow-up of aplastic anaemia. To further elucidate the molecular basis of PNH occurring in childhood, we used the powerful and rapid protein truncation test to scan for truncative mutations in the entire PIG-A mRNA reverse transcribed and amplified from blood mononuclear cells. The somatic defect responsible for PNH in the patient was found to be a splicing mutation, IVS5+1G→A, which has previously been described in two Asiatic adults with PNH.