Detection of late pp67-mRNA by NASBA in peripheral blood for the diagnosis of human cytomegalovirus disease in AIDS patients.

OBJECTIVE: To evaluate the value of late-pp67-mRNA nucleic acid sequence-based amplification (NASBA) in comparison to DNA-PCR, blood culture and pp65-antigenemia assay for the detection of human cytomegalovirus (HCMV) disease in HIV-infected patients. METHODS: The results of pp67-mRNA NASBA, DNA-PCR, culture and pp65-antigenemia assay were compared in 402 whole blood specimens of 98 HIV-infected patients with a low CD4 lymphocyte count who had not yet received highly active antiretroviral therapy (HAART). Thirty-seven samples were obtained from 30 patients with a diagnosis of HCMV disease and 365 samples from 68 patients without HCMV disease. RESULTS: The highest agreement of test results was observed between pp67-mRNA NASBA and quantitative pp65-antigenemia, with a threshold of nine antigen-positive cells/10(5) leukocytes (kappa-value 0.70, 95% CI=0.58-0.82). The sensitivity of pp67-mRNA NASBA for the diagnosis of HCMV disease (59.3%) was identical to that of the quantitative pp65-antigenemia assay, higher than that of the blood culture (48.2%) but lower than that of the DNA-PCR (77.8%). Pp67-mRNA NASBA (92.3%), quantitative pp65-antigenemia assay (92.3%) and blood culture (93.9%) were highly specific for the diagnosis of HCMV disease and as a result, had a higher positive predictive value (76.2, 76.2 and 76.5%, respectively) than the qualitative DNA-PCR (58.3%) and the qualitative pp65-antigenemia assay (47.6%). CONCLUSION: pp67-mRNA NASBA, an easy and rapid to perform assay, well-standardised by virtue of co-amplified internal system control RNA, provides a high specificity and positive predictive value for the diagnosis of HCMV disease in HIV-infected patients, comparable to that of the quantitative pp65-antigenemia assay and blood culture.

Value of different assays for detection of human cytomegalovirus (HCMV) in predicting the development of HCMV disease in human immunodeficiency virus-infected patients.

In the present prospective study, five blood tests for detection of human cytomegalovirus (HCMV), nucleic acid sequence-based amplification (NASBA) for detection of early (immediate-early antigen) and late (pp67) mRNA, PCR for detection of HCMV DNA (DNA PCR), culture, and pp65 antigenemia assay, and culture and DNA PCR of urine and throat swab specimens were compared for their abilities to predict the development of disease caused by HCMV (HCMV disease). Of 101 human immunodeficiency virus (HIV)-infected patients with

At home management of aplastic phase following high-dose chemotherapy with stem-cell rescue for hematological and non-hematological malignancies.

BACKGROUND: After high-dose chemotherapy with autologous stem-cell support long hospital stays in the aplastic phase are expensive, lead to increased risk of hospital infections and to increasing pressure on available hospital beds. We developed a home care regimen that allows patients to be at home for most of the aplastic period, without daily hospital visits. PATIENTS AND METHODS: Between October 1995 and December 1997, transfer of supportive care to the home setting took place in three phases for patients undergoing high-dose chemotherapy with stem-cell transplant for malignant lymphoma (one course of BEAM), breast cancer or germ-cell cancer (three courses of tCTC). In the inpatient cohort, the supportive care designed for at home use was administered in the hospital until neutrophile recovery to 0.5 x 10(9)/l. In the second, outpatient cohort, patients were discharged the day after stem-cell reinfusion but the supportive care was delivered daily in hospital. The third, home care cohort, consisted of patients who were discharged the day after stemcell reinfusion, after which specialized home care professionals delivered all supportive care including transfusions and parenteral antibiotics at home, with once weekly check-up in hospital by the transplant physician. RESULTS: Forty-two patients were treated with 81 cycles of high-dose chemotherapy (11, 18 and 13 patients and 17, 40 and 24 courses in the inpatient, outpatient and home care cohorts respectively). Inpatients were hospitalized in the aplastic phase for a median of 14 days. Patients in the outpatient cohort were at home in the aplastic phase for a median of six days (with a median of six days in hospital), and in the home care cohort for a median of 10 days (with a median of 1.5 days in hospital). Unscheduled readmissions and hospital visits were frequent in the outpatient and home care cohorts, mostly due to fever, central indwelling catheter malfunctioning or chemotherapy-related toxicity. However, patients could usually be discharged again after observation and treatment. No infectious deaths or unexpected emergencies occurred in the outpatient or home care cohort. Neither was there any suggestion of an increased number of fevers, infections, or other complications. CONCLUSIONS: At home management in the aplastic phase after high-dose chemotherapy and stemcell transplant by community-based professionals is feasible without signs of increased toxicity or infections.

Quantitative pp65-antigenemia assay for the prediction of human cytomegalovirus disease in HIV-infected patients.

OBJECTIVE: To evaluate the ability of a quantified pp65-antigenemia assay to predict the development of human cytomegalovirus (HCMV) disease in patients with an advanced HIV infection. DESIGN: A prospective longitudinal study between March 1993 and December 1996. Blood samples for the pp65-antigenemia assay were drawn at 2-3 month intervals. SETTING: AIDS department of an institutional tertiary care centre. PATIENTS: A total of 101 HIV-infected patients with CD4 lymphocyte counts of 100/mm3 or less were enrolled. Ninety-seven patients were eligible for analysis. All patients gave informed consent. MAIN OUTCOME MEASURES: The development of HCMV disease. RESULTS: Of the 97 patients, 24 developed HCMV disease after a median follow-up of 10.6 months. Three months before the development of HCMV disease, an increase in the median number of pp65-antigen-positive leukocytes was observed. The highest combination of sensitivity (45%) and specificity (94%) for the development of HCMV disease within the next 3 months was found when an assay cut-off level of 48/10(5) pp65-antigen-positive leukocytes was applied, with a positive predictive value (PPV) for the development of HCMV disease of 75%. The Kaplan-Meier estimate of HCMV disease-free survival after patients reached 48/10(5) or more antigen-positive leukocytes on longitudinal follow-up was a median 3.7 months [95% confidence interval (CI), 2.5-8.5]. The hazard ratio (HR) of this threshold level for the development of HCMV disease was 9.6 (95% CI, 4.2-21.8). CONCLUSION: Longitudinal follow-up using the pp65-antigenemia assay of HIV-infected patients with a low CD4 lymphocyte count improves the identification of patients who will develop HCMV disease in the foreseeable future, and should be considered for the selection of patients who may benefit from pre-emptive HCMV treatment.

Hepatitis C virus infections in dialysis centers in The Netherlands: a national survey by serological and molecular methods.

A national survey of hepatitis C virus (HCV) infections among dialysis patients in The Netherlands was performed. The study involved 2,653 patients (2,108 hemodialysis patients and 545 chronic ambulatory peritoneal dialysis [CAPD] patients) from 39 of the 49 dialysis centers in the country. Patient sera were analyzed by both serological and molecular methods. Screening by a third-generation enzyme immunoassay (EIA) yielded 79 reactive sera. The presence of anti-HCV antibodies was confirmed in 70 patients by a line immunoassay. All seropositive samples were tested by reverse transcriptase PCR, and 57 samples were found to contain HCV RNA. Of the nine EIA-positive and line immunoassay-negative or indeterminate samples, four were HCV RNA positive. All seronegative samples were screened for the presence of HCV RNA in pools of five sera. Of 2,576 antibody-negative samples, 6 contained HCV RNA. All antibody-positive and RNA-positive samples were also tested by a second serological assay. The prevalence of HCV infections among Dutch dialysis patients as determined by serology or the presence of HCV RNA was 3% (80 of 2,653), i.e., 3.5% (73 of 2,108) in patients treated on hemodialysis and 1.3% (7 of 545) in patients on CAPD. Of these 80 HCV-infected dialysis patients, 67 (84%) were HCV RNA positive. Serological screening alone would have diagnosed only 70 infected patients. Therefore, antibody screening combined with detection of HCV RNA should be considered as the "gold standard" for diagnosing HCV infection in dialysis patients. The prevalence of HCV-infected patients in Dutch dialysis centers ranged from 0 to 8%, suggesting the existence of local risk factors for acquiring HCV infection. Genotyping analysis by reverse hybridization line probe assay revealed the presence of genotypes la (23%), 1b (46%), 2 (3%), 2a (13%), 2b (1%), 3a (7%), and 4a (4%). In four (6%) samples multiple genotypes were detected. The genotype distribution of HCV isolates among Dutch dialysis patients was similar to the distribution among nondialysis patients from the Benelux, except for subtype 1a, which was significantly more prevalent among dialysis patients. In only one center, a high prevalence of an uncommon genotype was suggestive of infection from a common source.

Risk factors for HCV infection in two haemodialysis units in The Netherlands.

BACKGROUND: In order to assess risk factors for HCV infection during haemodialysis, all patients receiving haemodialysis for more than 6 months in two separate units in the Netherlands were studied retrospectively. METHODS: Antibodies to HCV, HCV-RNA and HCV genotypes were determined. Risk factors were identified by analysis of an extensive collection of clinical data. RESULTS: In unit A, 8 out of 75 (11%) patients and in unit B 4 out of 122 (3%) patients had antibodies to HCV. Eleven out of the 12 anti-HCV-positive patients had detectable HCV-RNA. Genotyping showed the presence of 4 different genotypes in unit A (1, 1a, 2b, and 3a). Three patients in unit B were infected with the same genotype (1b), where one of these patients was also infected with genotype 1a. One patient in unit B did not have detectable HCV-RNA. The risk of acquiring a HCV infection in unit A was associated with the number of blood transfusions. However, in unit B this risk was associated with the duration of dialysis. Other factors such as the number of surgical procedures were not associated with HCV infection. CONCLUSIONS: Blood transfusions and the dialysis process itself are important and independent risk factors for HCV transmission in dialysis patients. Surgical events do not appear to be important risk factors. However, relative risks may vary considerably between different dialysis centres.

Comparative in-vitro activity of meropenem against selected pathogens from hospitalized patients in The Netherlands. MASTIN Study Group.

Thirty laboratories evaluated the in-vitro activity of meropenem and 15 commonly used antibiotics against selected microorganisms isolated in 1994-95 from hospitalized patients with serious infections requiring antibacterial treatment. Isolates (2169) from blood, sputum, pus or CSF were included. MICs were determined with Etest and NCCLS breakpoints were used. In general, the MICs of meropenem for Gram-positive isolates were found to be one- to six-fold higher than those of imipenem, except for Enterococcus faecalis. The MIC90 of meropenem for E. faecalis was high (32 mg/L) and distinctly higher than the MIC90 of imipenem (2 mg/L). The MICs of meropenem for Gram-negative isolates were two- to 24-fold lower, with the exception of Acinetobacter spp. Gram-negative fermentative strains, Enterobacter spp. in particular, isolated from patients in intensive care units (ICU) were more resistant to the -lactam antibiotics than those isolated from patients in non-intensive care wards. However, all Enterobacteriaceae, with and without inducible -lactamases, isolated from ICU patients were susceptible to meropenem.

Gastric non-Hodgkin lymphomas of mucosa-associated lymphoid tissue are not associated with more aggressive Helicobacter pylori strains as identified by CagA.

Primary gastric non-Hodgkin lymphoma of mucosa-associated lymphoid tissue (MALT-NHL) is strongly related to Helicobacter pylori (H pylori) infection. CagA positive H pylori strains appear to be more aggressive, as reflected by a greater degree of gastric inflammation and higher levels of Il-8 production. Using a polymerase chain reaction-based assay, cagA status was determined in H pylori strains of 12 patients with gastric MALT-NHL, 38 patients with peptic ulcer disease, and 39 patients with non-ulcer dyspepsia. CagA-positive H pylori strains were significantly more frequent in peptic ulcer disease than in non-ulcer dyspepsia (Fisher's exact test p <0.001). An increased frequency of CagA-positive strains was not identified in gastric MALT-NHL compared to non-ulcer dyspepsia patients (Fisher's exact test P <.3). Therefore, it can be concluded that infection with more aggressive strains is associated with the development of peptic ulcer disease, but plays a minor role, if any, in the development of gastric MALT-NHL.

[A measles epidemic in an adequately vaccinated middle school population]. / Een mazelenepidemie in een goed gevaccineerde middelbare-schoolpopulatie.

OBJECTIVE: To assess the extent of a measles epidemic in a secondary school. DESIGN: Retrospective and questionnaire investigation. SETTING: Secondary school, Bilthoven. METHOD: Questionnaire followed by laboratory testing for measles and other infectious diseases with exanthema. RESULTS: The response rate was 99% (935/949 pupils, aged 12-21 years, vaccination rate 92%). Seventy-seven students underwent laboratory investigations. Measles virus was isolated in 2 suspected patients. Thirty-three of 37 patients with clinical or laboratory criteria of measles had been vaccinated. Complications of measles were not detected. Infection was also detected in patients with relatively few or atypical symptoms. The protective efficacy of measles vaccine could be determined because the attack rate of the school population was less than 5%. CONCLUSION: Primary failure of the measles vaccine might be the cause of the minor epidemic but the results do not cast doubt on the efficacy of the current measles vaccination programme.

Prevalence of antibodies to hepatitis C virus in a Dutch group of haemodialysis patients related to risk factors.

In January 1992, 122 chronic haemodialysis patients (> 9 months dialysis) from the University Hospital Utrecht and its outpatient dialysis facility were tested for the presence of anti-hepatitis C virus (HCV) antibodies. The objective was to identify risk factors for HCV infection in chronic haemodialysis patients in an attempt to explain the high prevalence of anti-HCV antibodies found among such haemodialysis patients. A second generation enzyme linked immuno-sorbent assay (EIA) was used as a screening test. Results were confirmed by a recombinant immunoblot assay and by the polymerase chain reaction (PCR). Four (3.3%) of 122 patients reacted positively in the EIA screening test as well as in the immunoblot assay; three of these were positive using PCR. None of the patients with anti-HCV antibodies had received blood products other than blood from transfusions, none had markers for a hepatitis B virus (HBV) infection or admitted intravenous drug abuse. A total number of 2395 units of blood, unscreened for HCV, had been administered to our dialysis group, an average of 19.6 (SD 44.7) units per patient. The seroprevalence of anti-HCV antibodies among blood donors in Utrecht was 0.03%. Patients with antibodies to HCV had been on dialysis longer than those dialysis patients without HCV antibodies (odds ratio 1.8, 95% CI 0.99-3.29). We conclude that the risk for HCV infection for this dialysis group can only partially be attributed to unscreened blood transfusions. Haemodialysis itself may play a role in transmission of HCV.

A randomized study on the effect of bladder irrigation with povidone-iodine before removal of an indwelling catheter.

We have evaluated the effect of povidone-iodine (PVP-I) bladder irrigation prior to catheter removal on subsequent bacteriuria. Of 264 patients, 138 received PVP-I irrigation and 126 were controls. Both groups were similar with respect to duration of catheterization and bacteriuria before removal of the catheter. Of 497 cultures taken after catheter removal 99 (20%) were positive. This included 52 of 233 in the control group (22%) and 47 of 264 in the study group (18%). Patients with positive cultures had a significantly longer period of catheterization. Our trial did not demonstrate that PVP-I bladder irrigation before catheter removal reduces subsequent bacteriuria.

Detection of Chlamydia trachomatis in clinical specimens by the polymerase chain reaction.

Sequences derived from the endogenous plasmid of Chlamydia trachomatis and from the genes coding for ribosomal 16S RNA of Chlamydia psittaci were used as primers and oligonucleotide probes for detection of chlamydiae by the polymerase chain reaction. The endogenous plasmid primers generated specific amplified products of 517 bp with all known Chlamydia trachomatis serovars. No specific products of Chlamydia psittaci and Chlamydia pneumoniae could be detected using these primers. With the rRNA primers specific amplified products of 208 bp were generated with Chlamydia psittaci, Chlamydia trachomatis and Chlamydia pneumoniae. No specific amplified products were detected with DNA isolated from a variety of microorganisms from the urogenital and the respiratory tract. Of 156 clinical specimens used for evaluation of the polymerase chain reaction, 26 were found to be positive for Chlamydia trachomatis on culture. All 26 culture positive samples were also found to be positive for Chlamydia trachomatis DNA by the polymerase chain reaction with both primer sets. Two culture negative samples were also found to be positive by this technique. The polymerase chain reaction thus seems to be a sensitive and reliable method for detection of Chlamydia trachomatis.

Serodiagnosis of respiratory syncytial virus (RSV) infection in children as measured by detection of RSV-specific immunoglobulins G, M, and A with enzyme-linked immunosorbent assay.

The diagnostic value of an enzyme-linked immunosorbent assay for detection of respiratory syncytial virus (RSV)-specific immunoglobulin G (IgG), IgM, and IgA in sera from infants and children with proven RSV infection, from a control group, and from patients with symptoms of viral respiratory disease was analyzed. Compared to virus isolation and RSV antigen detection methods, the sensitivity of this assay was 87% and the specificity was 79%. For IgG alone, these were 45 and 92%, for IgM alone they were 48 and 92%, and for IgA alone they were 74 and 95%, respectively.

Favourable effect of detergent on antigen detection and comparison of enzyme linked detection systems in an ELISA for Chlamydia trachomatis.

The influence of addition of detergents to the antigen on sensitivity of an ELISA for the detection of Chlamydia trachomatis was investigated. Of the detergents tested, only octyl-beta-d-glucopyranoside and sodiumdesoxycholate gave respectively a two- to fourfold and an eightfold increase in sensitivity. The effect was only present within a narrow range of detergent concentrations. The optimal detergent concentration was strongly dependent on the protein concentration in the antigen preparation. For optimal detection of the bound chlamydial antigen, enzyme and biotin labeled secondary antibodies were compared. The biotin labeled antibodies were combined with enzyme labeled streptavidin-biotin complex (SBC). Color development was obtained with both types of conjugates by using either o-phenylenediamine (OPD) or an enzyme amplification system. The best results were obtained with the SBC method and OPD.

Human serum antibody response in Campylobacter jejuni enteritis as measured by enzyme-linked immunosorbent assay.

An ELISA for detection of IgG, IgA, and IgM antibody using an acid-glycine extract from Campylobacter jejuni as antigen was developed. To determine the value of this assay for the diagnosis of acute Campylobacter jejuni infections, the IgG, IgA, and IgM immune response against Campylobacter jejuni was investigated at various timepoints after infection in patients with culture-proven infection. A total of 112 sera from 46 patients and 78 sera from a control group were tested. All but one of the 46 patients with culture-proven Campylobacter jejuni enteritis developed IgG antibodies against Campylobacter jejuni. IgA and IgM ELISA both showed 97% specificity, and sensitivity of 63% and 30% respectively. IgG antibody titers generally remained at a constant level for more than 50 days, whereas IgA and IgM antibody titers declined more rapidly to normal values within 30 to 50 days after onset of clinical symptoms. Detection of Campylobacter jejuni specific IgA antibodies in a single serum sample provided the most useful assay for serological diagnosis of Campylobacter jejuni enteritis. The presence of Campylobacter jejuni specific IgM antibodies was the sole diagnostic criterion in three cases. Serological diagnosis of Campylobacter jejuni enteritis should therefore include both IgA and IgM antibody determination.

Interlaboratory evaluation of indirect enzyme-linked immunosorbent assay, antibody capture enzyme-linked immunosorbent assay, and immunoblotting for detection of immunoglobulin M antibodies to Toxoplasma gondii.

One hundred and fifteen serum samples from healthy laboratory personnel and 50 consecutive samples from 19 patients with anamnestic clinical signs of toxoplasmosis were assayed by four laboratories for the presence of immunoglobulin M antibodies to Toxoplasma gondii by an indirect enzyme-linked immunosorbent assay (ELISA), an antibody capture assay with peroxidase-labeled toxoplasma antigen, and an immunoblotting assay. In addition, a commercially available antibody capture ELISA was used. Highly significant correlation coefficients were obtained between the four laboratories and the commercial test. The indirect ELISA and antibody capture ELISA showed equal sensitivity in detection of immunoglobulin M antibodies to toxoplasma in early-stage serum samples. However, in this study, the antibody capture assay discriminated better between serum samples obtained at early or late stages of toxoplasma infection.