Diagnosis of chronic disseminated candidosis from liver biopsies by a novel PCR in patients with haematological malignancies.

Hepatic Candida infection (HCI; known as chronic disseminated candidosis or CDC) is a distinct form of disseminated Candida infection with predominant involvement of the liver. Diagnosis of HCI is usually made on clinical suspicion together with multiple lesions in liver on ultrasound (US), CT and/or MRI scan. Fungal elements may not always be visible in liver tissue and mycological culture is frequently negative, making the evidence for proven fungal disease difficult. We studied a novel commercially available low-cost and density-array (LCD) chip technique for a molecular diagnosis of HCI. This is a two-step procedure with PCR amplification after DNA extraction followed by hybridization on a small chip provided by the manufacturer (Fungi 2.1, Chipron GmbH). The analysis of DNA from 45 fungal control strains showed an excellent specificity and sensitivity. The DNA from 11 liver biopsies of patients with haematological malignancies suffering from CDC was analysed on the LCD chip and overall 11 fungal pathogens could be detected in eight liver biopsies, supporting the clinical diagnosis of HCI/CDC. Analysis of liver biopsies from controls was negative for fungal DNA in all samples studied. In conclusion, the novel LCD chip technique examined in our study was able to detect fungal pathogens in liver biopsies from patients with haematological malignancies and suspected HCI/CDC but was negative in control biopsies.

Parvovirus B19 infection associated with unilateral cervical lymphadenopathy, apoptotic sinus histiocytosis, and prolonged fatigue.

This report describes the case of a 16 year old girl with a history of high fever, prolonged fatigue, and cervical lymphadenopathy of the right side. In addition, the patient showed neutropenia, thrombopenia, and pronounced reticulopenia. Cervical ultrasound showed unilateral hypoechoic lymph nodes up to 23 mm in diameter suspicious for malignant lymphoma. Histology of a cervical lymph node specimen revealed massive nodular histiocytic proliferation and prominent apoptosis without necrosis. Parvovirus B19 was detected by polymerase chain reaction and immunohistochemistry in the lymph node. In summary, this case is an unusual presentation of parvovirus B19 infection. The virus was identified as the potential causative agent of unilateral cervical lymphoma and apoptotic sinus histocytosis, thus broadening the clinicopathological spectrum of parvovirus B19 induced diseases.

Treatment of acute hepatitis C infection in HIV-infected patients: a retrospective analysis of eleven cases.

Studies on hepatitis C virus (HCV) monoinfected patients suggest high sustained treatment response rates of up to 98% when interferon monotherapy is administered during the acute phase of HCV-infection. To clarify whether early treatment of acute hepatitis C is similarly efficient in human immunodeficiency virus (HIV) positive patients, we conducted a retrospective survey of HIV-positive patients with acute HCV infection. Eleven HIV-positive patients who had been treated with interferon or interferon/ribavirin were identified at eight HIV-specialty outpatient clinics. The patients had been treated over a median 25 weeks with standard interferon (two patients), pegylated interferon (four patients) and pegylated interferon in combination with ribavirin (five patients). A post-treatment response (negative serum HCV-RNA at the end of treatment) was seen in 10 of 11 patients and HCV-RNA remained undetectable 24 weeks after the end of treatment in all the 10 responders. Alanine aminotransferase (ALT) normalized in eight patients while two virological responders and one nonresponder showed persistent mild ALT elevations. In conclusion, early treatment of acute hepatitis C seems to achieve high sustained virological treatment response rates also in patients with HIV-infection.

[Management of primary HIV-infection]. / Umstrittenes Management der primären HIV-Infektion. lm Frühstadium antiretroviral behandeln--ja oder nein?

Clinical suspicion of a primary HIV infection is aroused by the patient's exposure history and can only be confirmed by an HIV PCR test. The classic HIV antibody test (ELISA) often is non-reactive during the very early stage of HIV infection. The value of antiviral therapy during primary HIV infection is unclear. In spite of a positive influence on surrogate markers in vitro, studies so far have not been able to demonstrate a clinical benefit. Treatment should be carried out only in specialized infectious disease centers, most preferably within controlled clinical studies.

Fatal atypical T-cell proliferation associated with Epstein-Barr virus infection.

We report the case of a young Caucasian man who presented with polyneuropathy and severe, ultimately fatal, congestive heart failure in the context of a chronic active Epstein-Barr virus (EBV) infection. Post-mortem examination revealed both monoclonal and polyclonal proliferation of EBV-positive atypical T lymphocytes within different organs. Predominant infiltration of the nervous system and heart with extensive myocardial scarring accounted for the clinical symptoms. The remarkable features of this case are (i) the occurrence in a Caucasian patient, (ii) the absence of detectable immunodeficiency, and (iii) the myocardial destruction by EBV-infected monoclonal T cells.

Mycobacterium microti llama-type infection presenting as pulmonary tuberculosis in a human immunodeficiency virus-positive patient.

A rare case of Mycobacterium microti infection in a human immunodeficiency virus-positive patient is described. Because of unusual morphological and cultural features, the pathogen was analyzed by spoligotyping and identified as the Mycobacterium microti llama type. Although culture of M. microti is difficult, drug susceptibility testing could be performed, which correlated with the clinical outcome.

Telomerase activity in cervical smears.

It is well known that almost all carcinoma cells including those of the uterine cervix have re-established their telomerase activity. However, until now there is no conclusive picture on the telomerase activity in cervical dysplasias and about their relationship to HPV infection. To investigate this question, material from 34 patients (15 with normal epithelium, 11 with LGSIL, 8 with HGSIL) obtained by conventional cervical brushing was used and subjected to non-radioactive TRAP-ELISA (Boehringer Mannheim). The HPV analysis was performed by PCR on formalin-fixed, paraffin-embedded biopsy material obtained after cytological investigation. We could show that telomerase activity is detectable in normal cervical epithelium, and that an gradual increase exists for both telomerase activity and HPV positivity from normal epithelium to HGSIL. However, HPV infection and telomerase activity appear to be independent of each other. The high frequency of telomerase positivity in patients with normal cervical epithelium indicates that telomerase activity is not a useful differential diagnostic aid. Whether patients with telomerase-positive dysplasias have a higher probability to progress into an invasive carcinoma remains to be clarified by follow-up studies.

Chromosomal imbalances in brain metastases of solid tumors.

Metastases account for approximately 50% of the malignant tumors in the brain. In order to identify structural alterations that are associated with tumor dissemination into the central nervous system we used Comparative Genomic Hybridization (CGH) to investigate 42 brain metastases and 3 primary tumors of 40 patients. The metastases originated from lung cancer (14 cases), melanomas (7), carcinomas of breast (5), colon (5), kidney (5), adrenal gland (1) and thyroid (1). In addition, tumors of initially unknown primaries were assessed in 3 cases. The highest incidence of DNA gains were observed for the chromosomal regions 1q23, 8q24, 17q24-q25, 20q13 (>80% of cases) followed by the gain on 7p12 (77%). DNA losses were slightly less frequent with 4q22, 4q26, 5q21, 9p21 being affected in at least 70% of the cases followed by deletions at 17p12, 4q32q34, 10q21, 10q23-q24 and 18q21-q22 in 67.5% of cases. Two unusual narrow regional peaks were observed for the gain on 17q24-q25 and loss on 17p12. The incidence at individual loci can be viewed at our CGH online tumor database at http:// amba.charite.de/cgh/. The metastases of each tumor type showed a recurrent pattern of changes. In those cases with primary tumor and metastases available, the CGH pattern exhibited a high degree of conformity. In conclusion, our data suggests that specific genetic lesions are associated with tumor dissemination into the nervous system and that CGH analysis may be a useful supplementary tool for classification of metastases with unknown origin.

[PCR based diagnosis in pathology]. / Zur PCR-basierten Erregerdiagnostik in der Pathologie.

PCR-based diagnosis of infectious diseases has developed into an important method in surgical pathology. We summarize our experience in this area based on 3500 analyses performed during the past 5 years. The peculiarity of material from a pathological laboratory is the risk of DNA fragmentation as a consequence of tissue fixation. For this reason the assays used must be adapted to this particular situation. PCR-based diagnosis of infectious diseases, if performed according to appropriate quality standards, is a safe and effective technique with high sensitivity and specificity and results in an etiologically based diagnosis.

Human papilloma virus status and chromosomal imbalances in primary cervical carcinomas and tumour cell lines.

Human papilloma virus (HPV) infection is the crucial step in the initiation of cervical carcinomas. In addition, HPV18 has been implicated in tumour progression and adverse clinical outcome. We determined the HPV types in 12 primary cervical carcinomas and 12 cell lines and compared the findings with the comparative genetic hybridisation (CGH) pattern of chromosomal alterations. The most frequent alteration was the deletion at 3p14 followed by the loss of 2q34-q36 along with 3q gain. High risk HPV types were detected in all samples except one primary tumour. In contrast to the normal distribution, HPV18 was present in 75% of cases including all cell lines. The cell lines carried a higher number of genetic alterations and a different CGH pattern for several chromosomes than the primary tumours, despite microdissection. Purely HPV18 positive cases indicated a high incidence of imbalances at specific loci with peaks of the histogram coinciding with known HPV integration sites. The study suggests that HPV infection is associated with a recurrent pattern of chromosomal changes in cervical carcinomas and that the development and progression of these alterations is triggered by integration into the host genome.

Delayed low density lipoprotein (LDL) catabolism despite a functional intact LDL-apolipoprotein B particle and LDL-receptor in a subject with clinical homozygous familial hypercholesterolemia.

We identified a 38-yr-old male patient with the clinical expression of homozygous familial hypercholesterolemia presenting as severe coronary artery disease, tendon and skin xanthomas, arcus lipoides, and joint pain. The genetic trait seems to be autosomal recessive. Interestingly, serum concentrations of cholesterol responded well to diet and statins. We had no evidence of an abnormal low density lipoprotein (LDL)-apolipoprotein B (apoB) particle, which was isolated from the patient using the U937 proliferation assay as a functional test of the LDL-binding capacity. The apoB 3500 and apoB 3531 defects were ruled out by PCR. In addition, we found no evidence for a defect within the LDL-receptor by skin fibroblast analysis, linkage analysis, single-strand conformational polymorphism and Southern blot screening across the entire LDL-receptor gene. The in vivo kinetics of radioiodinated LDL-apoB were evaluated in the proband and three normal controls, subsequently. The LDL-apoB isolated from the patient showed a normal catabolism, confirming an intact LDL particle. In contrast the fractional catabolic rate (d-1) of autologous LDL in the subject and the normal controls revealed a remarkable delayed catabolism of the patient's LDL (0.15 vs. 0.33-0.43 d-1). In addition, the elevation of LDL-cholesterol in the patient resulted from an increased production rate with 22.8 mg/kg per day vs. 12.7-15.7 mg/kg per day. These data indicate that there is another catabolic defect beyond the apoB and LDL-receptor gene causing familial hypercholesterolemia.

Analysis of novel apolipoprotein B mutations using a modified U937 cell line LDL binding assay.

The U937 myelomonocyte proliferation assay can be used to detect patients with familial defective apolipoprotein B-100 (FDB). Previous studies have employed electronic cell counting to assess cell proliferation. We simplified the assay using 3H-thymidine incorporation DNA analysis to measure cell growth. We tested the modified method by analyzing the effects of different concentrations of native low density lipoproteins (LDL), methylated LDL, as well as LDLs obtained from patients with FDB on cell growth. Methylation of LDL to various degrees reduced cell proliferation correspondingly, and LDLs obtained from FDB patients decreased cell growth confirming that the modified method was able to detect binding defective species of LDL. We applied this method to analyze three novel apoB polymorphisms recently characterized in this laboratory (apoB His1896-->Arg, apoB Asn1887-->Ser, apoB Ala4454-->Thr), which did not significantly alter U937 cell proliferation. Our results show that this simplified assay can be used for screening for LDL variants with defective binding.

Rapidly progressive seronegative spondylarthropathy with atlantodental subluxation in a patient with moderately advanced HIV infection.

It has been suggested that the immunosuppressed state associated with HIV infection may influence the clinical course of rheumatic diseases. We describe the case of a patient with moderately advanced HIV infection who developed a psoriatic rash and a rapidly progressive spondylarthropathy of the cervical spine with atlantodental subluxation requiring spondylodesis. This case supports the hypothesis that HIV infection may be associated with uncommon manifestations and a rapidly progressive course refractory to medical therapy in patients with spondylarthropathy.

Influence of selective management on the prognosis and the risk of rupture of abdominal aortic aneurysms.

A total of 199 patients with abdominal aortic aneurysms were followed up to investigate the influence of selective management on the prognosis and the risk rupture of abdominal aortic aneurysms. Decisions to operate or to continue watchful waiting with treatment of risk factors for expansion were based on aneurysm size, expansion rate, aneurysm-related symptoms, and individual operative risk. Rupture occurred in eight cases. All aneurysms were larger than 5 cm, and six were larger than 6 cm in diameter at the last measurement before rupture. The resulting overall 5-year cumulative rate of rupture was 7.3% (Kaplan Meier). The 134 patients who underwent more than one ultrasound examination were observed for an average of 4.0 years (536 patient-years). The expansion rate was significantly correlated with the initial diameter and the diastolic blood pressure (best subset multiple regression analysis: r = 0.403; P < 0.001). A correlation with the systolic blood pressure was found only in univariate analysis (r = 0.236; P = 0.011). Amplitude of blood pressure, serum cholesterol level, low-density and high-density lipoproteins, ratio of low- to high-density lipoproteins age, and the extent of smoking habits were not correlated with the expansion rate. Our conclusion is that larger diameter and higher diastolic blood pressure are important risk factors for expansion of abdominal aortic aneurysms. Selective management of abdominal aortic aneurysms based on aneurysmal size, expansion rate, and patient characteristics may result in a low rate of rupture.

Strong inhibition of mammalian lipoxygenases by the antiinflammatory seleno-organic compound ebselen in the absence of glutathione.

Both human recombinant 5-lipoxygenase (EC 1.13.11.34) and 15-lipoxygenase (EC 1.13.11.33, mammalian enzyme) purified from rabbit reticulocytes were inhibited in the absence of glutathione (GSH) by submicromolar concentrations of the seleno-organic compound ebselen. These concentrations were comparable to those of the enzymes. Soybean lipoxygenase-1 (EC 1.13.11.33, plant enzyme) was not inhibited, whereas prostaglandin endoperoxide synthase-1 (EC 1.14.99.1) was inhibited only at much higher concentrations of ebselen (IC50 = 37.7 +/- 4.3 microM). The action of ebselen on reticulocyte 15-lipoxygenase (IC50 = 0.17 +/- 0.01 microM) was studied in detail. Inhibition occurred instantaneously and appeared to be reversible and was largely abolished by a 20-fold molar excess of GSH over ebselen. In the presence of 1 mM GSH 50% inhibition was observed only at ebselen concentrations as high as 234 +/- 27 microM. 13S-hydroperoxy-9Z, 11E-octadecadienoic acid, the lipoxygenase product formed from linoleic acid, augmented the inhibitory effect at low concentrations and caused a partial reversal at high concentrations. A variety of derivatives or structural analogues of ebselen were also tested and proved to be either inactive or weaker inhibitors of 15-lipoxygenase. We have concluded that the potent inhibition of 15-lipoxygenase by ebselen is due neither to GSH peroxidase-like activity nor to lowering of the hydroperoxide tone. The pharmacological implications of these unique characteristics of the action of ebselen on lipoxygenases are then discussed.

Inhibitory effects of sulfonated shale oils (ammonium bituminosulphonates, Ichthyols) on enzymes of polyenoic fatty acid metabolism.

The two commercial pharmaceutical preparations of ammonium bituminosulphonates, Leukichthol and Dark Ichthyol, were shown to inhibit the formation of 5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HETE) from external arachidonic acid by human polymorphonuclear leukocytes stimulated by ionophore A-23187 in a dose-dependent manner. Pure arachidonate 15-lipoxygenases from rabbit reticulocytes and soya beans, and the particulate prostaglandin endoperoxide synthase from sheep vesicular glands, were also inhibited. With the reticulocyte lipoxygenase, the Ichthyols suppressed the enzyme activity by two different mechanisms: (1) a prolongation of the lag period typical of lipoxygenase catalysis, and (2) by a lowering of the maximal enzymatic activity after the end of lag period. As expected, the first effect was reversed by the addition of the lipoxygenase product 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE). Ammonium bituminosulphonates are thus universal inhibitors of lipoxygenase activities, and the latter are of potential importance in inflammatory dermatoses.