Aymptomatic CMV viremia is associated with increased levels of serum amyloid A in patients with advanced HIV-infection.

OBJECTIVE: We evaluated assays for the measurement of acute phase protein levels in plasma for their usefulness to identify sensitively an inflammatory response to active cytomegalovirus CMV infection in HIV-infected patients. METHODS: Plasma samples were collected from 28 CMV-seropositive patients with advanced HIV-infection (CD4-cell count <200/microl) before commencement of antiretroviral therapy. Sensitivity, specificity, and area under receiver operating characteristic curve for the selected acute phase protein assays (haptoglobin, fibronectin, high-sensitivity C-reactive protein (hs-CRP), human interleukin-6, serum amyloid A (SAA), and human lipopolysacharide binding protein) were compared with results of a CMV-specific PCR assay. RESULTS: CMV viremia was detectable in 8/28 patients. Levels of SAA correlated well with those of hs-CRP (r' = 0.439, P = 0.019 (Spearman rank correlation)). Levels of SAA >3 mg/L discriminated with 100% sensitivity and 40% specificity between HIV-infected patients with and without active CMV infection. Sensitivity of fibronectin was 100% and specificity 15% at a threshold-value corresponding with the lower limit of normal values as defined by the manufacturer of the assay (>29 mg/dL). Levels of the other acute phase proteins evaluated did not correlate with detection of CMV-DNA in plasma. CONCLUSION: Increased levels of SAA indicate sensitively an inflammatory response to active CMV infection. Use of a CMV-specific virological assay is required to confirm the specificity of a high SAA-level but may be limited to samples with high SAA-levels. Hence, screening for increased levels of SAA in patients with advanced HIV-infection may allow early identification of active CMV infection.

Asymptomatic HIV infection is characterized by rapid turnover of HIV RNA in plasma and lymph nodes but not of latently infected lymph-node CD4+ T cells.

OBJECTIVES: To study the kinetics of plasma viraemia and HIV-infected lymph-node cells in stable asymptomatic HIV infection with high CD4+ T-cell counts. METHODS: Nine asymptomatic HIV-infected patients with stable CD4+ T-cell counts (510-1350 x 10(6)/l) were treated with a triple-drug combination. Plasma viraemia was determined at days 0, 3, 7, 10, 14, 21 and 28 of treatment [Roche polymerase chain reaction (PCR) and ultrasensitive PCR assay]. Sequential lymph-node biopsies were examined in four patients before and after 4 weeks of treatment. Productively infected cells were counted in lymph-node sections (in situ hybridization). The infection rates of FACS-sorted CD4+ lymph-node T cells and the expression of single-spliced, double-spliced and full-length HIV transcripts were determined. RESULTS: HIV plasma RNA half-lives ranged from 1.4 to 2.7 days. Viral turnover varied between 0.07 and 7.54 x 10(8) copies per day. The number of productively infected lymph-node cells as well as the amount of extracellular virus in germinal centres was markedly reduced during treatment, paralleled by a clearance of single-spliced, double-spliced and full-length HIV transcripts from CD4+ lymph-node T cells. Plasma viraemia remained detectable with an ultrasensitive PCR assay in three out of four patients. The percentage of lymph-node CD4+ T cells harbouring proviral DNA decreased only slightly. CONCLUSIONS: The kinetics of HIV replication are rapid in stable asymptomatic infection, and the magnitude of replication varies considerably. Productively infected lymph-node cells and extracellular virus in germinal centres undergo a rapid turnover, whereas latently infected CD4+ T cells have a lower rate of turnover. The latter may contribute substantially to viral persistence during therapy.

Comparison of three HCV genotyping assays: a serological method as a reliable and inexpensive alternative to PCR based assays.

BACKGROUND: Determination of hepatitis C virus (HCV) genotypes and subtypes is of rising clinical importance. In times where also an increasing need for cost effectiveness can be observed, the demand for fast and easy performable assays grows. OBJECTIVES: To evaluate and compare different genotyping methods regarding their reliability, practicability, and expense in the daily routine. METHODS: Sera of 39 patients infected with different HCV subtypes were examined by a serological genotyping assay (NS-4 IBA), by the widely used INNO-LiPA HCV II, and by a nucleotide sequencing method. RESULTS: The tests performed equally well in terms of HCV subtyping and no different results were obtained. However, the serotyping assay provided the results in less than half the time needed by the other two assays. Significant differences were also observed regarding the 'hands on' times and the costs. The technical equipment which was necessary to perform the assays is significantly reduced using the serological assay. CONCLUSION: Our study demonstrates that the serological test offers the opportunity to determine HCV genotypes and subtypes reliably, fast, easy, and cost effective.

High rate of chronicity in HCV infection determined by antibody confirmatory assay and PCR in 4110 patients during long-term follow-up.

BACKGROUND: It is still unclear how many patients with hepatitis C virus (HCV) antibodies have viremia and hence are infectious. OBJECTIVES: To determine the chronicity of HCV infection by correlation of HCV antibodies with presence of viremia in long-term follow-up. STUDY DESIGN: In a longitudinal study sera of 4110 patients were analyzed with second generation HCV-enzyme immunoassay (EIA) and polymerase chain reaction (PCR). Only those patients were included in this study in whom sequential serum samples over a period of 2 years were available. To avoid preanalytical and analytical failures, we used a transport solution to prevent RNA degradation and a four-antigen recombinant immunoblot assay, established in our laboratory, for confirmation of antibody reactivity. RESULTS: Of 2815 patients with confirmed HCV antibodies 2784 (98.9%) were also positive in HCV-PCR assay. False reactive EIA results were detected in 177 (13.7%) individuals as shown by confirmatory assay and PCR. Only one patient (0.04%) spontaneously lost detectable HCV viremia and subsequently HCV-specific antibodies. CONCLUSIONS: Our study clearly demonstrates that presence of confirmed HCV-specific antibodies correlates significantly (98.9%; P < 0.001) with HCV viremia, and that spontaneous loss of viremia is a very rare event in HCV infection. We also found that elimination of HCV infection is not sufficiently predicted by the loss of detectable viremia in PCR, but can be concluded from the disappearance of virus-specific antibodies.

Application of HIV-1 genotypic-resistance testing prevents the evolution of further resistance mutations in heavily pretreated patients.

BACKGROUND: Resistance-associated mutations in HIV-1 evolve even under highly active antiretroviral therapy. OBJECTIVE: To evaluate the clinical efficacy of genotypic-resistance testing (GRT), to estimate the potential of a given antiretroviral therapy for prevention of further resistance mutations. STUDY DESIGN: Ten patients were treated prospectively with drugs, according to the results of a GRT. Five patients were allocated to group I in which antiretroviral therapy could be switched to an effective regimen (consisting of at least three sensitive drugs, from at least two different classes of antiretroviral substances). Five patients (group II) had no option for effective therapy, and continued to be treated non-effectively (at least one applicated substance class only intermediately sensitive, or resistant). GRT and quantitative viral cultures were performed longitudinally for 8 months. Also, plasma HIV-1 RNA, total CD4+ cells, and rates of productively infected CD4+ cells were determined. RESULTS: All the patients in group I showed a significant decrease of HIV-RNA of >1 log/ml (mean, -1.35 log/ml, P=0.025). The mean increase of CD4+ cells was 46 (not significant). The rate of productively infected CD4+ cells decreased significantly (mean, -16 productively infected CD4+ cells per 10(6) total CD4+ cells, P=0.04). In this group no further resistance mutations were detected after 8 months. In group II, none of the patients showed a significant decrease of HIV-1 RNA (mean, +0.05 log/ml), total CD4+ cells decreased (mean, -35, not significant), the rate of productively infected CD4+ cells increased significantly (mean, +124 productively infected CD4+ cells per 10(6) total CD4+ cells, P=0.04), and 4 of 5 patients had additional mutations in the RT gene conferring multi-drug resistance within 8 months (P=0.048). CONCLUSIONS: GRT is predictive of the efficacy of a therapeutic regimen, in particular regarding evolution of further resistance mutations.

In vivo dynamics and pathogenicity of wild-type and resistant Hepatitis B virus during long-term lamivudine monotherapy - a clinical note.

BACKGROUND: Genotypic resistance of Hepatitis B virus (HBV) against lamivudine evolves within months after onset of therapy. OBJECTIVES: To determine the longitudinal order in which resistance mutations appear and to compare the kinetics and pathogenicity of wild-type and resistant HBV. STUDY DESIGN: In a longitudinal study, consecutive samples were drawn over a period of 28 months from a patient with chronic hepatitis B, and resistance mutations were followed by sequencing a part of the polymerase region of HBV. These data were compared with HBV copy numbers, HBsAg and ALT levels, and results of consecutive liver biopsies. RESULTS: After 21 weeks of treatment, a silent mutation at codon 528 (CTG to TTG) occurred. Significant genotypic resistance was detectable after 68 weeks, indicated by a substitution of isoleucine for methionine at residue 552 (M552I). Nineteen weeks later, the virus exhibited additional resistance-associated mutations (L528M and I552V). The resulting high-level resistance was reflected by an increase of serum HBV copies of 4.7 log(10). The turnover of wild-type and resistant HBV was 2.6x10(6) and 1.8x10(6) virions/day, respectively. HBsAg and ALT levels were lower within the period when resistant HBV was detectable. During treatment the progress of liver fibrosis was arrested. CONCLUSIONS: The in vivo replicative capacities and dynamics of wild-type and resistant HBV were similar. However, resistant HBV seemed to exhibit reduced pathogenicity.

Lior-serotype variants in Campylobacter isolates from the same stool sample.

Campylobacter strains isolated from the same stool sample were characterised by determination of biochemical properties and both heat-labile (Lior) and heat-stable (Lauwers) serotypes. In six of 60 campylobacter-infected stools, two or three strains differing in Lior-serotype were isolated from the same stool. In four of these six cases, the isolates with different Lior-serotypes showed identical biochemical reactions and identical heat-stable antigenic patterns. A predominant Lior-serotype was not detected among them but Lauwers-antigens O:3, O:14 and O:16 were found in isolates from three of the six stool samples. Moreover, the isolates were identified as C. coli in 76.5% of the stool samples (p < 0.05). We believe that variation in heat-labile antigens occurs in vivo and might be associated particularly with certain heat-stable serotypes of C. coli.

Comparison of conventional autoradiography with a new DNA enzyme immunoassay for the detection of hepatitis C virus-polymerase chain reaction amplification products.

The detection of HCV-PCR amplification products by DNA enzyme immunoassay (DEIA) was compared with conventional hybridization carried out with a 32P-labelled oligonucleotide probe. The detection limit of both methods was shown to be between 100 pg and 1 ng of amplicon. All serum samples of 40 HCV-seropositive patients were positive after PCR in autoradiography, but only 38 with the DEIA technique (sensitivity 95%). There were no false-positive reactions by either method. The advantage of the DEIA method was the fast and non-radioactive detection of HCV amplicons. DEIA combines the specificity of the hybridization event with the speed of an ELISA procedure and is suitable for HCV-PCR.

Prognostic significance of the hepatitis B virus-DNA concentration in patients after orthotopic liver transplantation.

Hepatitis B virus-DNA was detected by polymerase chain reaction in 9 out of 10 patients after orthotopic liver transplantation. Three of these patients were at the same time positive for hepatitis B virus-DNA by dot-blot hybridization (hepatitis B virus-DNA > 1.5 pg/ml). In these three patients HBs-antigen (HBsAg) reappeared within a mean time of 12 weeks after orthotopic liver transplantation (range 7-18 weeks). Only two of the six polymerase chain reaction-positive and dot-blot-negative patients (hepatitis B virus-DNA between 0.4 fg/ml and 1.5 pg/ml) had recurrence of HBsAg within a mean time of 54 weeks (range 52-56 weeks). Passive immunoprophylaxis with anti-HBs antibodies (serum titers > 100 IU/l) did not prevent infection of the graft in the five reinfected patients. We conclude that a low concentration of serum hepatitis B virus-DNA after orthotopic liver transplantation, which is detectable only by polymerase chain reaction, indicates a delayed infection of the graft.

Evaluation of the results of delayed rhinoplasty in cleft lip and palate patients. Functional and aesthetic implications and factors that affect successful nasal repair.

Patients born with cleft lip and palate (CLP) present with a variety of nasal deformities. These are either congenital or iatrogenic. Our aim was to establish a correlation between aesthetic and functional nasal impairments in patients with CLP whose nasal reconstruction had been delayed. Fifty-four randomly selected patients with CLP deformities, all of whom had delayed nasal repairs were evaluated objectively, aesthetically in three planes, and functionally for symptoms of nasal obstruction, chronic maxillary sinusitis, and olfactory disturbances. Aesthetically the patients were analysed from 1:1 life-size full face, profile, and submental-vertex photographs, and full skull cephalograms. Nasal patency was assessed by rhinomanometry. The presence of chronic maxillary sinusitis and olfactory disturbances were deduced from the history. The degree of nasal dismorphism correlated with the severity of nasal functional impairments. Delayed nasal repairs in patients with CLP did not produce satisfactory aesthetic or functional results, probably because growth was retarded and midfacial development was disturbed at the time of delayed rhinoplasty and resulted in asymmetry. In CLP the nose should be repaired during the early primary cheilorhinoplasty, as this is essential for the restoration of a normally functioning and aesthetically pleasing nose.

Fatal influenza A infection with Staphylococcus aureus superinfection in a 49-year-old woman presenting as sudden death.

A fatal case of influenza A infection with Staphylococcus aureus superinfection in a previously healthy 49-year-old woman presenting as sudden, unexpected death is reported. Autopsy revealed severe necrotizing tracheobronchitis and hemorrhagic pneumonia. Microscopic examination of the trachea and bronchi showed mucosal necrosis and a dense lympho-monocytic infiltration of all layers. The lungs showed focal hemorrhagic pneumonia. No pathological changes were detectable in the myocardium. Influenza A virus was detected in bronchi and lung samples obtained during autopsy by the polymerase chain reaction (PCR) and bacterial superinfection with Staphylococcus aureus was shown by culturing from tracheal, bronchial and pulmonary swabs obtained during autopsy. PCR assays for the detection of Panton-Valentine leukocidin performed from all samples were negative. This case demonstrates the need for an interdisciplinary approach towards an organism-specific diagnosis of potentially infection-related deaths undergoing a medico-legal autopsy. With improved diagnostic possibilities such as PCR and DNA sequencing, forensic pathologists can, in close association with the field of microbiology, make a significant contribution to the detection of highly infectious agents which must be notified to the authorities. This will increase particularly the knowledge about the influence of these agents on sudden, unexpected deaths in outpatients.

Prevalence, incidence, and clinical relevance of the reverse transcriptase V207I mutation outside the YMDD motif of the hepatitis B virus polymerase during lamivudine therapy.

The reverse transcriptase V207I mutation within the hepatitis B virus (HBV) polymerase is associated with resistance to lamivudine in vitro. The prevalence of this mutation in treatment-naive patients was 1% (1/96). A follow-up of the patient carrying this mutation prior to treatment revealed no loss of sensitivity of HBV to lamivudine in vivo.

Hepatitis C virus infection in pregnancy and the risk of mother-to-child transmission.

The risk of vertical transmission of the hepatitis C virus (HCV) from infected mothers to their children during pregnancy and delivery was determined in 120 children born to HCV-positive mothers. Methods included enzyme immunoassay and immunoblot for detection of HCV antibodies and reverse transcription polymerase chain reaction (RT-PCR) for detection of viral RNA. Six (5%) children were perinatally infected with HCV as shown by RT-PCR. None of the infected children had clinical signs of hepatitis. None of the pregnancies was complicated by abortion, stillbirth, premature birth, or malformation of the child. Special concern was given to the possibility of HCV transmission via breast milk. In no breast milk sample obtained from 34 HCV-infected mothers was HCV RNA detected. These observations indicate that HCV infection is not necessarily a contraindication for breast-feeding.

Study on reliability of commercially available hepatitis C virus antibody tests.

The serodiagnosis of hepatitis C virus (HCV) infection was analyzed by a recombinant immunoblot assay (RIBA) with recombinant proteins encoded by the viral RNA isolated from our patients in Hamburg, Germany. The HCV RNA was amplified by PCR, and proteins encoded by the viral core and the NS3, NS4, and NS5 regions were expressed subsequently in Escherichia coli. The results obtained with our UKE RIBA were compared with the results of the Abbott HCV second-generation enzyme immunoassay (EIA). Serum samples from 270 patients, which were sent to us on the suspicion of HCV hepatitis and which were negative for hepatitis A virus and hepatitis B virus antibodies, were examined. In 227 cases (84.1%), there were identical positive (204 cases, 75.6%) or negative (23 cases, 8.5%) results in both tests. In 32 cases (11.9%), the reactive Abbott second-generation HCV EIA results could not be confirmed by the UKE RIBA and the HCV PCR. In follow-up studies conducted over 1 year, these results did not change. In three cases (1.1%), the UKE RIBA presented a positive result while the Abbott second-generation HCV EIA was negative. All three cases were positive in the HCV PCR and showed seroconversion in an HCV EIA 4 to 6 weeks later. In addition, 33 patient serum samples were examined by UKE RIBA in parallel with the Ortho RIBA 2.0. In three cases (9.1%), a positive Ortho RIBA 2.0 result could not be confirmed by the UKE RIBA and the HCV PCR. All three patients were free of complaints. The UKE RIBA showed also a smaller number of indeterminate results (3.0%) than the Ortho RIBA 2.0 (24.2%). This comparison study demonstrates that the commercially available HCV antibody tests should be further improved.