[Maxillary sinus infection with emphysema of the head as a "red herring" in steroid associated colon perforation]. / Kieferhöhleninfekt mit Kopfemphysem als "red herring" bei Steroid-assoziierter Kolonperforation.

Steroid therapy increases the risk of bowel perforation. Bowel perforation may occur at any time of steroid therapy, but the first weeks appear to hold the greatest potential for perforation. However, clinical findings after perforation may be misleading under steroids, and peritonitis may be absent. It is known that bowel perforation can lead to subcutaneous emphysema at various sites. Thus, in any patient with emphysema, bowel perforation must be included in the differential diagnosis, especially in patients receiving steroids. Missing knowledge of this entity may lead to marked delay between onset of initial signs and diagnosis, and hence worsen the survival rate. In this report we present a case of chronic steroid use, where asymptomatic sigma perforation led to a generalized emphysema, which was initially attributed to a maxillary sinus infection due to Aspergillus and anaerobic bacteria.

Resistance analyses in HIV infected patients with a history of multiple antiretroviral treatment regimens.

OBJECTIVE: To assess HIV-1 isolate based resistance profiles from extensively pretreated patients and effects of a resistance guided switch of antiretroviral therapy. METHODS: In a prospective study phenotypic and genotypic resistance analyses were performed on HIV infected individuals with failure of the current therapy and history of at least three antiretroviral regimens. Antiretroviral therapy was changed according to the results. Viral load and CD4 lymphocyte counts were measured at baseline, after 10 (SD 2), and 24 (2) weeks. RESULTS: All patients (n=52) failed their actual regimen. Currently versus ever previously taking the specific drug, resistance associated mutations and phenotypic resistance to AZT and 3TC were found in over 80% of individuals; resistance to DDI and D4T was detected in less than 10% of cases. A resistance guided switch of therapy was followed by a median decrease of viral load of 0.5 log10 units after 24 weeks. Individuals resistant to two or more drugs compared with patients with resistance to less than two drugs of ongoing treatment, were switched to a regimen containing DDI, D4T, and a PI or NNRTI. After 10 (SD 2) weeks viral load decrease was pronounced in patients with resistance to at least two drugs in the previous regimen. CONCLUSIONS: Among different RTI, the profile of clinically relevant resistance indicates pronounced differences when looking at separate drugs. Regarding virological response, in the context of available drugs, resistance tested with currently used methods is of limited value in extensively pretreated patients and seems to have its value primarily in first or second switch of therapy.

Multicenter study of a commercial, automated polymerase chain reaction system for the rapid detection of Mycobacterium tuberculosis in respiratory specimens in routine clinical practice.

A cooperative study was conducted among six laboratories to compare the performance of the Cobas Amplicor (CA) polymerase chain reaction (PCR) system (Roche Molecular Systems, USA) for the detection of Mycobacterium tuberculosis with that of microscopy and culture in routine clinical laboratory diagnosis. A total of 5,221 decontaminated respiratory specimens were tested. The use of an internal control allowed detection of PCR inhibition in 144 (2.8%) specimens. Only two culture-positive samples were CA PCR inhibitory and therefore could not be detected by PCR testing. Of the 333 culture-positive specimens, 278 (83.5%) were positive by the CA PCR. Of the 4,744 culture-negative specimens, 52 (1.1%) were positive by the CA PCR. After analysis of discrepancies, 40 of the 52 culture-negative, CA PCR-positive specimens were classified as true positive. Thus, the overall sensitivities of culture, CA PCR and microscopy were 89.3%, 85.2% and 55.5%, respectively. The overall specificity of the CA PCR was 99.7%. Five of the six centers found similar performances for the CA PCR, with sensitivities ranging from 85.7 to 90.9%. The CA PCR was more sensitive for smear-positive samples, exhibiting overall sensitivities of 96.1% and 71.7% for smear-positive and smear-negative specimens, respectively. These results indicate that the Cobas Amplicor system enables microbiology laboratories with reasonable previous experience in molecular biology testing to perform PCR and to detect Mycobacterium tuberculosis in more than 70% of specimens obtained from infected patients.

In vitro analyses of diamond-like carbon coated stents. Reduction of metal ion release, platelet activation, and thrombogenicity.

Coronary artery stents can induce platelet activation by shear forces, contact to the biomaterial, and release of metal ions. This activation is one important trigger for thrombosis. Coating of stents is a possible approach to prevent this side effect. The purpose of this study was to evaluate in vitro the biocompatibility of stents coated with diamond-like carbon (DLC). Semiquantitative energy-dispersive X-ray microanalyses showed a complete coverage of the DLC stents. Flow cytometric analyses revealed a significantly higher increase of mean channel fluorescence intensity for the activation-dependent antigens CD62p and CD63 in non-coated compared to DLC-coated stents (p<0.05). Atomic adsorption spectrophotometry analyses revealed a significant release of nickel and chromium metal ions by non-coated stents over a storage period of 96 hours in human plasma (p<0.05). In contrast, only minimal concentrations of released ions could be detected in the case of DLC-coated stents. Similar observations were made with inductively coupled plasma mass spectrometry analyses. Here, high concentrations of molybdenum and manganese ions were released from non-coated stents (p<0.05), while release of these ions from DLC-coated stents was virtually undetectable (p=0.1 for molybdenum and p=0.4 for manganese). Coating of intracoronary stents with diamond-like carbon significantly improves biocompatibility. This biocompatible coating may therefore contribute to a reduction in thrombogenicity.

The role of antibiotic prophylaxis in the treatment of acute pancreatitis.

Infected necrosis in acute pancreatitis is one of the most dreaded complications of acute pancreatitis. Whereas selection of an appropriate antibiotic treatment of the infection poses no problem, prophylactic application of antibiotic remains controversial in the absence of symptoms of infection, but where contrast-enhanced CT scan clearly proves necrosis. This article discusses the present state of the art of the role of antibiotic prophylaxis in the treatment of acute pancreatitis and provides clinical guidelines.

Chlamydia trachomatis and male infertility: chlamydia-IgA antibodies in seminal plasma are C. trachomatis specific and associated with an inflammatory response.

BACKGROUND: There is controversy over the role of asymptomatic genital tract infection by Chlamydia trachomatis, its optimal diagnosis, and its place in the etiology of male infertility. OBJECTIVE: Comparison of direct detection of Chlamydia trachomatis in semen with the presence of chlamydia-antibodies in seminal plasma and serum, together with parameters of the spermatogram, in men of infertile relationships. STUDY DESIGN: Prospective clinical study. SETTING: University hospital tertiary referral center. SUBJECTS AND METHODS: Two groups of consecutive andrological patients (n = 89 and n = 36) were investigated as follows: semen analysis, including concentration of granulocyte-elastase; detection of C. trachomatis in semen samples and first void urine by polymerase chain reaction (PCR) and antigen-ELISA (Celisa); detection of chlamydia antibodies in serum and seminal plasma by recombinant antibody-enzyme-linked immunosorbent assay (rELISA) and of Chlamydia trachomatis specific antibodies by the ImmunoComb-Chlamydia-Bivalent test. RESULTS: In 2/125 (1.6%) semen samples Chlamydia trachomatis DNA was detected by PCR. Genus specific anti-chlamydia-IgA was found in 12/122 (9%) of the seminal plasmas. This IgA appeared to be specific for C. trachomatis. Seminal plasmas with chlamydia-IgA antibodies showed higher PMN-elastase levels than IgA negative samples (P < 0.04). Chlamydia-IgG antibodies were present in 27/89 (30%) of the sera, but in only five of these 27 sera (19%) were the antibodies detected specific for C. trachomatis. There were no associations between any of these variables and the parameters of the routine semen analysis. CONCLUSION: IgA-chlamydial antibodies in seminal plasma appeared to be specific against C. trachomatis and were associated with an inflammatory response in the male genital tract.

Presence of the major outer-membrane protein of Chlamydia trachomatis in patients with chronic salpingitis and salpingitis isthmica nodosa with tubal occlusion.

OBJECTIVE: To determine the presence of the major outer-membrane protein of Chlamydia trachomatis in fallopian tube tissue specimens of infertile women with chronic salpingitis and/or salpingitis isthmica nodosa with tubal occlusion. DESIGN: Prospective controlled study. SETTING: Department of Obstetrics and Gynecology, University of Bochum, Herne, Germany. PATIENT(S): Fifty-six consecutive infertile women with histologically documented chronic salpingitis and/or salpingitis isthmica nodosa and bilateral tubal occlusions were evaluated. They were compared with 28 fertile women. INTERVENTION(S): Fallopian tube tissue specimens were taken during reconstructive infertility surgery, including cesarean section and tubal ligation. MAIN OUTCOME MEASURE(S): Detection of the major outer-membrane protein of C. trachomatis in fallopian tube tissue specimens by a direct fluorescent antibody test. RESULT(S): The major outer-membrane protein of C. trachomatis was found in fallopian tube tissue specimens in 11 of 56 infertile patients (20%) with chronic salpingitis and/or salpingitis isthmica nodosa. The median titer of IgG serum antibodies to Chlamydia was significantly higher in women with the major outer-membrane protein of C. trachomatis than in patients without this antigen. In comparison, the major outer-membrane protein of C. trachomatis was not found in any of the fallopian tube tissue specimens of the control group. CONCLUSION(S): The presence of the major outer-membrane protein of C. trachomatis is associated with chronic salpingitis and/or salpingitis isthmica nodosa with tubal occlusion.

Multicenter evaluation of the COBAS AMPLICOR HCV assay, an integrated PCR system for rapid detection of hepatitis C virus RNA in the diagnostic laboratory.

The benefits shown by the recent introduction of PCR for the in vitro diagnosis of hepatitis C virus (HCV) infection has prompted the development of standardized, ready-to-use assays that can be implemented in routine clinical laboratories. We have evaluated the clinical performance of COBAS AMPLICOR HCV (COBAS), the first instrument system that allows the automation of HCV RNA amplification and detection, to determine its performance in the routine laboratory setting. More than 2,000 specimens collected at five centers were analyzed in parallel by the COBAS and the manual AMPLICOR HCV (AMPLICOR) tests, and the results were compared with the results for biochemical and serological markers of HCV. In this study the two PCR systems showed the same accuracy, with a concordance rate of 99.8%. As expected, the correlation between serology and PCR was not absolute because the presence of anti-HCV antibodies may be associated with a latent or past infection. On the other hand, if the presence of confirmed anti-HCV antibodies and elevated alanine aminotransferase levels are taken as the "gold standard," indicating an active, ongoing infection, the COBAS and AMPLICOR tests show high and comparable sensitivities (100%) and specificities (98%), with positive and negative predictive values of 100 and 97%, respectively. During the study no false-positive reactions were detected. The use of an internal control allowed the identification of inhibitory substances that prevented amplification for 0.3 and 0.4% of samples tested by the COBAS and AMPLICOR tests, respectively. Compared to the manual system, the COBAS system allowed a significant reduction of hands-on time and could improve the overall laboratory work flow. In conclusion, these results support the use of the COBAS and AMPLICOR tests for the molecular diagnosis of active HCV infections.

Species-specific identification of microsporidia in stool and intestinal biopsy specimens by the polymerase chain reaction.

In view of the increasing number of cases of human microsporidiosis, simple and rapid methods for clear identification of microsporidian parasites to the species level are required. In the present study, the polymerase chain reaction (PCR) was used for species-specific detection of Encephalitozoon cuniculi. Encephalitozoon hellem, Encephalitozoon (Septata) intestinalis, and Enterocytozoon bieneusi in both tissue and stool. Using stool specimens and intestinal biopsies of patients infected with Enterocytozoon bieneusi (n = 9), Encephalitozoon spp. (n = 2), and Encephalitozoon intestinalis (n = 1) as well as stool spiked with spores of Encephalitozoon cuniculi and Encephalitozoon hellem and tissue cultures of Encephalitozoon cuniculi and Encephalitozoon hellem, three procedures were developed to produce PCR-ready DNA directly from the samples. Specific detection of microsporidian pathogens was achieved in the first PCR. The subsequent nested PCR permitted species determination and verified the first PCR products. Without exception, the PCR assay confirmed electron microscopic detection of Enterocytozoon bieneusi and Encephalitozoon intestinalis in stool specimens and their corresponding biopsies and in spiked stool samples and tissue cultures infected with Encephalitozoon cuniculi and Encephalitozoon hellem. Moreover, identification of Encephalitozoon spp. could be specified as Encephalitozoon intestinalis. Whereas standard methods such as light and transmission electron microscopy may lack sensitivity or require more time and special equipment, the PCR procedure described facilitates species-specific identification of microsporidian parasites in stool, biopsies, and, probably, other samples in about five hours.

Rapid plasma virus and CD4+ T-cell turnover in HIV-1 infection: evidence for an only transient interruption by treatment.

OBJECTIVES: To analyse the short-term kinetics of viral plasma RNA and CD4+ T cells numbers in patients with different initial CD4+ T-cell counts treated with different antiretroviral regimens. METHODS: In 10 HIV-1 positive patients, in vivo kinetics of plasma HIV RNA and CD4+ T cells were studied during antiretroviral treatment. Lymphocyte subpopulation analysis, quantitative polymerase chain reaction (PCR), p24 antigen enzyme immunoassay (EIA) and beta 2-microglobulin EIA were performed at days 0, 3, 7, 10, 14, 21 and 28 of treatment. One additional patient served as a control. The resulting curves were fitted. Half-lives were calculated using the time constant T of decrease or increase [T1/2 = In(2) x T]. Calculations of virus and CD4+ T-cell turnover were multiplied by the total blood volume. RESULTS: Viral plasma RNA half-life ranged from 1.1 to 5.1 days, independent of prior or actual treatment and initial CD4+ T-cell count. The calculated peripheral blood viral plasma RNA turnover varied between 0.02 and 55.8 x 10(8) copies/ml/day and showed some negative correlation with initial CD4+ T-cell counts. CD4+ T-cell turnover estimates ranged from 0.01 to 7.5 x 10(8) cells/day. Most patients showed an immediate reincrease of virus load after the nadir. Changes in HIV p24 antigen paralleled HIV plasma RNA in p24 antigen-positive patients. beta 2-microglobulin decreased until day 7-15 in all but one case and rapidly reincreased to pretreatment values. CONCLUSIONS: The kinetics of virus and CD4+ T-cell turnover are uniformly rapid throughout a wide range of initial CD4+ T-cell counts. The magnitude of virus turnover varies considerably among individuals and appears to be inversely related to the initial CD4+ T-cell count. These data also argue for a rapid resumption of virus production and lymphocyte turnover during treatment.

An improved technique for the diagnosis of viral retinitis from samples of aqueous humor and vitreous.

We applied the technique of DNA amplification with the polymerase chain reaction to nine aqueous humor and five vitreous samples from HIV-1-infected patients with clinically diagnosed cytomegalovirus retinitis. For the amplification, recently published primers specific for herpes simplex virus (HSV), varicella zoster virus (VZV) and cytomegalovirus (CMV-1) were used. Additionally, a newly developed primer pair specific for the main immediately early gene of CMV (CMV-2) was selected and compared with the published one. All primers were tested on noninfected and HSV-, VZV- and CMV-infected human fibroblast cell culture supernatant, thereby excluding cross-reactivity of the chosen primers. In none of 13 aqueous humor and six vitreous samples of healthy controls was any viral DNA amplified. Using the CMV-1 primers, we detected CMV DNA in five of nine aqueous humor and three of five vitreous samples amplifying a DNA fragment 435 base pairs in length. With the CMV-2 primers, we detected a CMV DNA fragment with a length of 110 base pairs in eight of nine aqueous humor and in four of five vitreous samples. Additionally, CMV DNA was found in three of nine urine and two of nine saliva specimens. Both CMV and HSV DNA were amplified in one aqueous sample. Varicella DNA was not detected in any of the specimens. Thus, the polymerase chain reaction is more sensitive than other comparable diagnostic tests and may provide an alternative to conventional virus isolation and in situ hybridization techniques for the laboratory diagnosis of viral ocular disease.

Diagnosis of human cytomegalovirus-induced retinitis in human immunodeficiency virus type 1-infected subjects by using the polymerase chain reaction.

In 13 of 16 AIDS patients with retinitis, a herpesviruslike infection was diagnosed by clinical investigation. In 12 of the 13 patients, human cytomegalovirus (HCMV) DNA was detected in 5 microliters of aqueous humor by using the polymerase chain reaction (PCR). In the aqueous humor of 12 control patients HCMV DNA could not be detected by PCR. PCR may be used to monitor specific antiviral long-term therapy in HCMV retinitis.

Isolation of normal human follicular dendritic cells and CD4-independent in vitro infection by human immunodeficiency virus (HIV-1).

Immunohistological and electron microscopy studies of lymph nodes from patients infected with the human immunodeficiency virus 1 (HIV-1) demonstrated that follicular dendritic cells (FDC), the antigen-presenting cells of the B cell system, contain and may produce the virus. To elucidate the mode of infection of FDC with HIV-1 in vitro we developed an improved method for the preparation of single-cell suspensions of viable FDC with high purity (greater than 90% FDC). These isolated FDC were subjected to human T cell leukemia virus IIIB infection, which was monitored after 4 days in culture using the polymerase chain reaction. We were able to demonstrate that normal human FDC are highly susceptible to infection by HIV-1. Inhibition experiments with the monoclonal antibody OKT4a demonstrate that this infection is independent of the CD4 molecule.

Progression of HIV-1 infection. Monitoring of HIV-1 DNA in peripheral blood mononuclear cells by PCR.

We present data on the distribution of human immunodeficiency virus (HIV-1) proviral DNA in different subsets of peripheral blood mononuclear cells (PBMCs) over an observation period of eight months. Eleven patients with well documented HIV-1 infection were studied. The PBMCs were obtained at two intervals and purified by fluorescence-activated cell sorting (FACS) after staining with FITC-labelled monoclonal antibodies. Varying numbers of FACS-sorted CD4+ cells, CD8+ cells and peripheral monocytes were assayed for HIV-1 proviral DNA (env and gag region) by PCR. Samples from patients at CDC stages II or III had to contain 10(3)-10(4) cells in order to allow detection of proviral HIV-1 DNA. At CDC stage IV, however, HIV-1 DNA was detected in as few as 100 CD4+ T-lymphocytes. In contrast, in peripheral monocytes HIV-1 DNA was not regularly found. CD8+ cells did not harbor detectable amounts of proviral DNA. During an observation period of eight months, the rate of infected CD4+ T-lymphocytes increased significantly in three patients while staying constant in the remaining eight patients. This increase of the infection rate was paralleled by clinical progression in one patient and by a decrease of the absolute number of CD4+ cells in another patient. The percentage of CD4+ cells harboring the viral genome increases in the course of the disease. These results may help to explain the decrease in CD4+ T-lymphocyte counts during HIV-1 infection.

[Detection of HCMV-DNA in aqueous humor for diagnostic confirmation of viral retinitis]. / Nachweis von HCMV-DNA im Kammerwasser zur Diagnosesicherung der viralen Retinitis.

The differential diagnosis of viral retinitis is mainly based on the evaluation of the clinical findings by the ophthalmologist; for confirmation of the diagnosis, immunohistological testing is necessary. We present the method of polymerase chain reaction (PCR) as a new diagnostic tool in the examination of viral retinitis. The method is based on the selective amplification of a defined segment of viral DNA, which can in a second step be specifically detected by hybridization. Five patients with the typical clinical picture of a fresh cytomegaloviral retinitis of recent origin due to Aids and ten normal healthy controls were included in the study. We looked for CMV-genome in conjunctival and corneal epithelium, anterior chamber punctates, urine and sputum. Serum samples from the same patients were tested for CMV-IgM antibodies as sign of systemic immune response. They were all negative due to the systemic immune deficiency syndrome of these patients. We did not find CMV genome in any case in the control group or in any of the conjunctival and corneal epithelial samples, but we did find it in four of five anterior chamber punctates of patients with the clinical picture of CMV retinitis. We found CMV-DNA additionally in the urine of one patient and in urine and sputum of another. These results show the high value of the method of polymerase chain reaction in the diagnosis of uveitis.

CD34+ hematopoietic progenitor cells are not a major reservoir of the human immunodeficiency virus.

Hematologic abnormalities occur in the majority of patients with acquired immunodeficiency syndrome (AIDS). Infection of the hematopoietic progenitor cells has been proposed as a potential explanation. In this study, different bone marrow cell populations, including the CD34+ hematopoietic progenitor cells, were purified by a fluorescence-activated cell sorter (FACS) and analyzed for the presence of human immunodeficiency virus-1 (HIV-1) proviral DNA using the polymerase chain reaction. A group of 14 patients with AIDS or AIDS-related complex (ARC) was studied (11 with peripheral blood cytopenias). The CD4+ helper cells in the bone marrow were found positive for HIV-1 DNA in all patients. In contrast, CD34+ progenitor cells were positive in only one patient. Two monocyte samples and two samples of CD4-/CD34- lymphocytes/blasts (mainly B and CD8 lymphocytes) were positive. Proviral DNA could not be detected in granulocytes. FACS analysis showed that the percentage of CD34+ hematopoietic progenitor cells was not altered in the bone marrow of AIDS patients in comparison with the HIV-1 seronegative controls. In contrast, the number of CD4+ lymphocytes was markedly reduced in the bone marrow of AIDS patients. These results show that the hematologic abnormalities in AIDS patients are neither explained by direct infection of the hematopoietic progenitor cells with HIV-1 nor by a depletion of progenitor cells.