Prediction of severe disseminated adenovirus infection by serum PCR.

Adenoviruses are increasingly recognised as viral pathogens that can cause fatal infections in immunocompromised patients, particularly recipients of haematopoietic stem-cell grafts. Adenovirus infections are not easily diagnosed and the development of a severe infection cannot be predicted by standard culture techniques. In a pilot study, we investigated the value of adenovirus DNA detection in serum as a marker of disseminated disease in 14 patients with defined patterns of adenovirus infections. The results show that the appearance of adenoviral DNA in serum preceded the development of a severe or fatal adenovirus infection. Because proper management is dependent on early diagnosis and differentiation from other conditions, this test may be a valuable tool in the management of adenovirus infection.

PCR detection of adenovirus in a bone marrow transplant recipient: hemorrhagic cystitis as a presenting manifestation of disseminated disease.

Adenoviruses (AdV), causing fatal disseminated infections in bone marrow transplant (BMT) recipients, are associated not only with hemorrhagic cystitis (HC) but also with hepatitis, conjunctivitis, and viral interstitial pneumonia. The importance of this virus as a cause of disseminated disease, however, has remained underappreciated. AdV infection has been diagnosed primarily through the use of cell culture. The fact that cell culture is insensitive for detecting this virus has hindered recognition of the role that AdV may play in morbidity and mortality in BMT recipients. To emphasize these points, we describe a patient who presented with HC due to AdV serotype 11, genotype c, and died with disseminated infection. In addition to cell culture, this study used a newly developed PCR-based method, capable of detecting all AdV serotypes tested, including different genotypes of serotype 11. The PCR result was positive in all culture-positive samples, including samples of urine, conjunctiva, and bronchoalveolar lavage (BAL). Importantly, the PCR method provided evidence of urinary shedding of AdV in a pretransplant, culture-negative specimen and showed dissemination in a subset of culture-negative specimens, including BAL, blood, and bone marrow samples. The lack of widespread awareness of the fact that localized infections may presage dissemination, and the previous associated lack of rapid, sensitive diagnostic assays, has impaired recognition of AdV infections in patients undergoing BMT. Early detection may contribute to therapy modification and avoidance of unwarranted diagnostic procedures. It may also assist in epidemiologic control of this highly infectious pathogen and lead to a renewed interest in preventive and therapeutic approaches.

PCR method for detection of adenovirus in urine of healthy and human immunodeficiency virus-infected individuals.

Adenoviruses (AdV) cause diseases that range from localized, self-limited illnesses to fatal infections in immunocompromised patients. Culture is assumed to be sensitive but requires viable virus and up to 3 weeks for detection, and it can be inhibited by bacterial contamination. A new PCR method amplifying a region of the hexon gene was developed in order to detect AdV in urine more rapidly and with greater sensitivity than obtainable by culture technology. All 18 serotypes tested were detected. Quantitatively, with optimized urine processing, AdV PCR detected 0.2 PFU/ml (serotype 11) and 10 DNA copies/ml (serotype 2). Serially collected urine samples from human immunodeficiency virus (HIV)-infected patients with concurrent cytomegalovirus retinitis were divided into three groups: AdV culture-positive samples, AdV culture-negative or bacterially contaminated samples from patients with a history of AdV culture-positive urines, and AdV culture-negative samples from patients without a history of AdV culture positivity. Urine samples from healthy adults were also tested by culture and PCR to screen for asymptomatic shedding. Amplification was assessed with and without prior DNA purification. AdV was detected by PCR in 90% of culture-positive urines (100% of unclotted samples, e.g., those culture positive after storage for PCR testing), 71% of culture-negative or bacterially contaminated urines from AdV-infected patients, and 28% from AdV culture-negative patients. Healthy volunteers were culture negative for AdV, and 96% were PCR negative. The new AdV PCR method is rapid and sensitive and can detect viral DNA in samples for which culturing is problematic. The role of AdV replication during HIV infection merits further investigation with sensitive tools such as PCR.

Group B streptococcal colonization in a developing country: its association with sexually transmitted disease and socioeconomic factors.

Group B Streptococcus (GBS) is an important infectious organism in pregnant women and their neonates. Although excellent data are available from the developing world, little epidemiologic information is available from Latin America. To evaluate the prevalence of GBS colonization in a developing country, a prospective study was performed in Lima, Peru. We found a relatively low prevalence of GBS colonization of 6.0% in parturient women and 10.6% in nonpregnant women. No association of GBS colonization was made with previously identified risk factors such as age, parity, or birth control practices. We did find a positive association between GBS colonization and chlamydial carriage (P < 0.05). We also report an even distribution of GBS serotypes: Ia/c = 35%, IIc = 18%, III = 29%, and V = 18%. Our study provides evidence for a low prevalence of GBS maternal carriage in this urban Latin American population.

Learning and memory difficulties after environmental exposure to waterways containing toxin-producing Pfiesteria or Pfiesteria-like dinoflagellates.

BACKGROUND: At the beginning of autumn, 1996, fish with "punched-out" skin lesions and erratic behaviour associated with exposure to toxins produced by Pfiesteria piscicida or Pfiesteria-like dinoflagellate species were seen in the Pocomoke River and adjacent waterways on the eastern shore of the Chesapeake Bay in Maryland, USA. In August, 1997, fish kills associated with Pfiesteria occurred in these same areas. People who had had contact with affected waterways reported symptoms, including memory difficulties, which raises questions about the human-health impact of environmental exposure to Pfiesteria toxins. METHODS: We assessed 24 people who had been exposed. We collected data on exposure history and symptoms, did a complete medical and laboratory assessment (13 people), and carried out a neuropsychological screening battery. Performance on neuropsychological measures was compared with a matched control group. RESULTS: People with high exposure were significantly more likely than occupationally matched controls to complain of neuropsychological symptoms (including new or increased forgetfulness); headache; and skin lesions or a burning sensation of skin on contact with water. No consistent physical or laboratory abnormalities were found. However, exposed people had significantly reduced scores on the Rey Auditory Verbal Learning and Stroop Color-Word tests (indicative of difficulties with learning and higher cognitive function), and the Grooved Pegboard task. There was a dose-response effect with the lowest scores among people with the highest exposure. By 3-6 months after cessation of exposure, all those assessed had test scores that had returned to within normal ranges. INTERPRETATION: People with environmental exposure to waterways in which Pfiesteria toxins are present are at risk of developing a reversible clinical syndrome characterised by difficulties with learning and higher cognitive functions. Risk of illness is directly related to degree of exposure, with the most prominent symptoms and signs occurring among people with chronic daily exposure to affected waterways.

Viral resistance and CMV retinitis: design and methods of a prospective study. CRVR Research Groups. Cytomegalovirus Retinitis Viral Resistance Research Group.

A prospective study following a cohort of patients with newly diagnosed, previously untreated cytomegalovirus (CMV) retinitis is being conducted to study drug resistant CMV. Prior to initiation of treatment, patients undergo a baseline eye examination, fundus photography, and blood and urine culture for presence of CMV, and drug susceptibility testing against positive isolates. Patients are followed monthly with a detailed eye examination to diagnose progression of retinitis, and for fundus photography. Cultures are repeated at 1 and 3 months after enrollment, every 3 months thereafter, and at the time of treatment reinduction for the progression of retinitis. This study was designed to determine the prevalence and incidence of drug resistant CMV, as well as risk factors for the development of resistant CMV. It also will determine the correlation between clinical outcome, as measured both by eye examination and fundus photography, and viral resistance.

Cytomegalovirus retinitis and viral resistance. Prevalence of resistance at diagnosis, 1994. Cytomegalovirus Retinitis and Viral Resistance Study Group.

OBJECTIVE: To determine the prevalence of cytomegalovirus (CMV) isolates resistant to ganciclovir sodium or foscarnet sodium at the time of diagnosis of CMV retinitis, prior to the initiation of therapy. DESIGN: Prospective epidemiologic study. SETTING: An acquired immunodeficiency syndrome ophthalmology clinic. PATIENTS: Patients with acquired immunodeficiency syndrome and newly diagnosed CMV retinitis. INTERVENTION: Culturing blood and urine samples for CMV and testing of all positive isolates for sensitivity to ganciclovir and foscarnet. MAIN OUTCOME MEASURE: Prevalence of the following: blood culture isolates resistant to ganciclovir (inhibitory concentration 50% [IC50] > 5.5 mumol/L) or foscarnet (IC50 > 400 mumol/L) and urine culture isolates resistant to ganciclovir or foscarnet. RESULTS: Forty-nine patients were enrolled during a 13-month period. Forty-four patients had blood culture samples that could be evaluated; of these, 66% were positive (59% of patients). Thirty-four patients had urine cultures that were evaluable; of these, 82% were positive (57% of patients). Overall, 78% of patients had either a urine or blood culture sample positive for CMV. No blood culture isolates were resistant to ganciclovir, and only 1 urine culture isolate (2% of patients) was resistant to ganciclovir. Three percent of blood culture isolates and 4% of urine culture isolates (2% and 2% of patients, respectively) were resistant to foscarnet. Overall, 4% of patients had either a blood or urine culture isolate resistant to foscarnet. CONCLUSION: Resistance to ganciclovir or foscarnet at the time of diagnosis of CMV retinitis is uncommon.

Clostridium difficile colitis: an efficient clinical approach to diagnosis.

OBJECTIVE: To define clinical and laboratory variables that suggest the presence of Clostridium difficile colitis and to establish the number of stool specimens needed to reasonably exclude the diagnosis of C. difficile colitis. DESIGN: Prospective study of consecutive inpatients whose stool specimens were sent to be evaluated for the presence of C. difficile toxin. SETTING: University teaching hospital. PATIENTS: 268 hospital inpatients in medical, surgical, and gynecology units. MEASUREMENTS: Structured history and physical examination; detection of C. difficile toxin by cytotoxin tissue-culture assay with anti-C. difficile antiserum neutralization and by enzyme-linked immunoassay (EIA) for C. difficile toxins A and B; and detection of fecal leukocytes by microscopic examination and by latex agglutination lactoferrin assay. RESULTS: 43 of 268 consecutive inpatients were positive for C. difficile toxin by EIA or tissue-culture assay. Although toxin was detected by EIA alone in 39 of the 43 patients, it was detected in an additional 4 patients (10%) by tissue-culture assay alone. Univariate and multivariate logistic regression analysis showed that the following clinical and laboratory features were associated with C. difficile toxin positivity: the onset of diarrhea 6 or more days after the administration of antibiotics (odds ratio, 1.38 [95% CI, 1.10 to 3.79]); hospital stay longer than 15 days (odds ratio, 1.33 [CI, 1.09 to 3.95]); the presence of fecal leukocytes determined by microscopy (odds ratio, 2.39 [CI, 1.05 to 5.42]) or lactoferrin assay (odds ratio, 3.74 [CI, 1.80 to 7.76]); the presence of semiformed (as opposed to watery) stools (odds ratio, 2.33 [CI, 1.10 to 4.90]); and cephalosporin use (odds ratio, 2.36 [CI, 1.10 to 5.09]). Toxin-positive patients were no more likely than controls to have had fever, abdominal pain or cramps, leukocytosis, green-colored diarrhea, or blood in the stool or to have received clindamycin or penicillin derivatives. Of the 43 patients with C. difficile toxin, 34 (79%) had positive results for the toxin on the first stool specimen, 5 (cumulative, 91%) had positive results on the second specimen, and 4 had positive results on the third specimen. Overall, the negative predictive value of the first stool specimen was 97%. All patients who had two or more clinical or laboratory predictors were diagnosed with C. difficile disease when either the first or the second stool specimen was positive for toxin. CONCLUSIONS: Clinicians at the bedside can use readily available clinical and laboratory information to decide which patients are likely to have C. difficile disease and when it is appropriate and useful to order specific diagnostic tests for C. difficile toxin. Such data are also useful in determining the number of stool samples that reasonably excludes the diagnosis of C. difficile colitis.

Association of BK virus with failure of prophylaxis against hemorrhagic cystitis following bone marrow transplantation.

PURPOSE: Hemorrhagic cystitis (HC) after bone marrow transplantation (BMT) has been ascribed to cyclophosphamide metabolites. HC has also been associated with excretion of the BK type of polyomavirus. The relative contributions of cyclophosphamide metabolites and BK virus in the development of HC following BMT are unknown. PATIENTS AND METHODS: We conducted a randomized trial to compare mesna with forced diuresis for prophylaxis against HC in 147 BMT recipients. We studied the association of BK virus with HC in 95 consecutive BMT recipients by prospectively monitoring urinary excretion of BK virus using polymerase chain reaction amplification of viral gene sequences. RESULTS: HC occurred in 37 of 147 (25.2%) transplant recipients. The incidence of HC was similar in patients given mesna (26.8%, 19 of 71) or forced diuresis (23.7%, 18 of 76), and in recipients of allogeneic (27.2%, 18 of 64) or autologous marrow (22.9%, 19 of 83). The incidence of HC was unrelated to primary disease, preparative regimen, or occurrence of graft-versus-host disease (GVHD). Excretion of BK virus was demonstrated in 50 of 95 patients (52.6%); 38 patients (40%) had persistent BK viruria (> or = two consecutive positive samples). HC occurred in 19 of 38 patients (50%) with persistent BK viruria, in one of 12 (8.3%) with only a single urine sample positive for BK virus, and in none of 45 who did not excrete BK virus (P < .0001). Shedding of BK virus also had a strong temporal correlation with onset of HC (r = .95). CONCLUSION: Mesna and forced diuresis are equally effective in abrogating the urothelial toxicity of preparative regimens for BMT. Since HC after BMT is virtually always associated with persistent BK viruria, strategies aimed at the prevention or elimination of viruria in BK seropositive recipients are warranted.

Viral sensitivity testing in patients with cytomegalovirus retinitis clinically resistant to foscarnet or ganciclovir.

PURPOSE: Resistance to antiviral therapy is a potential cause of progression of cytomegalovirus retinitis in patients with the acquired immunodeficiency syndrome. We investigated the results of viral sensitivity testing in a series of patients with clinically resistant retinitis who had positive results of blood or urine cytomegalovirus cultures. METHODS: All patients with newly diagnosed cytomegalovirus retinitis between January 1990 and December 1991 were prospectively studied. Blood and urine cultures for cytomegalovirus were obtained in a nonrandomized subgroup of this group. The results of in vitro sensitivity to foscarnet and ganciclovir, determined by a DNA hybridization assay, were then analyzed in seven patients with clinically resistant cytomegalovirus retinitis and whose blood or urine culture results, or both, were positive for cytomegalovirus while on a treatment regimen. RESULTS: Foscarnet-resistant cytomegalovirus (ID50 > 300 microM) was isolated from two patients, one of whom was being treated with foscarnet. Ganciclovir-resistant cytomegalovirus (ID 50 > 6.0 microM) was isolated from four patients, three of whom were being treated with ganciclovir. Foscarnet- and ganciclovir-resistant cytomegalovirus occurred with previous ganciclovir therapy in one patient. Clinical improvement occurred in three patients whose change in therapy was based on viral sensitivity testing. In general, prolonged therapy with one drug was associated with a progressive increase in the ID 50 for that drug. CONCLUSIONS: Viral resistance to foscarnet or ganciclovir may explain refractory cytomegalovirus retinitis in some patients.

Comparison of four commercially available rapid enzyme immunoassays with cytotoxin assay for detection of Clostridium difficile toxin(s) from stool specimens.

Rapid (2.5- to 3.5-h) enzyme immunoassays (EIAs) for the detection of Clostridium difficile toxins have been developed. We report the results of simultaneous testing of 700 fresh stool specimens by the tissue culture cytotoxin assay and four EIAs (Bartels Prima System C. difficile Toxin A EIA, Cambridge Biotech Cytoclone A+B EIA, Meridian Diagnostics Premier C. difficile Toxin A EIA, and TechLab C. difficile Tox-A Test EIA). In cases of disagreement, culturing for toxigenic C. difficile was performed. A total of 61 (8.7%) specimens from 46 patients were positive for C. difficile toxin. The sensitivity of the cytotoxin assay was 87%, and that of culture was 93%. In comparison with the cytotoxin assay results, the sensitivity and specificity of the EIAs were as follows: Bartels, 87 and 96%; Cambridge, 89 and 99%; Meridian, 87 and 98%; and TechLab, 87 and 95%, respectively. In comparison with the cytotoxin assay plus toxigenic culture results, the sensitivity and specificity of the EIAs were as follows: Bartels, 84 and 97%; Cambridge, 85 and 99%; Meridian, 79 and 98%; and TechLab, 80 and 96%, respectively. The EIAs varied in positive predictive values (PPVs). A high PPV was seen with the Cambridge EIA (96%); lower PPVs were seen with the TechLab (64%), Bartels (72%), and Meridian (80%) EIAs because of high false-positive rates. The negative predictive values (98 to 99%) were excellent with all EIAs. Results were indeterminant with 0.3% of the samples by the Meridian EIA and 3% by all the other EIAs. Although the EIAs were less sensitive than the cytotoxin assay, they provide same-day results and may be useful in laboratories without tissue culture facilities.

In situ detection of lytic Epstein-Barr virus infection: expression of the NotI early gene and viral interleukin-10 late gene in clinical specimens.

Riboprobes that detect two genes expressed only during productive infection were developed to characterize the clinical spectrum of Epstein-Barr virus (EBV) lytic infection and identify diseases that may be responsive to antiviral drug therapy. The NotI antisense probe hybridizes to tandem repeats in the abundant early lytic cycle BHLF1 mRNA. Transcripts were detected in lytically infected cell lines, AIDS-associated oral hairy leukoplakia, bone marrow of a patient with virus-associated hemophagocytic syndrome, and spleen of an AIDS patient but not in EBV-positive primary central nervous system lymphomas or in circulating EBV-infected B cells from a patient with acute infectious mononucleosis. The viral (v) interleukin-10 (IL-10) probe hybridizes to the unique 5' end of the late lytic cycle BCRF1 mRNA, which encodes a protein homologous to the human cytokine IL-10. The vIL-10 probe detected transcripts in lytically infected cell lines and within the differentiated layers of oral hairy leukoplakia.

Rapid in situ hybridization for the diagnosis of latent Epstein-Barr virus infection.

Latent Epstein-Barr virus (EBV) infection is associated with a variety of malignancies. Rapid in situ hybridization techniques have been described for various lytic viral infections because of limited gene expression. However, EBERS (Epstein Barr early RNAs) are expressed in abundance in tumour cells which are latently infected with EBV. We have targeted these transcripts in a rapid (3 h) in situ hybridization assay fo the detection of latent EBV in clinical specimens, including formalin-fixed paraffin-embedded material. EBER RNA was detected in control cell lines which have two copies of the EBV genome and in paraffin-embedded biopsy specimens from patients with nasopharyngeal carcinoma, EBV-associated Hodgkin's disease, Burkitt's lymphoma and post-transplant lymphoma. The technique did not detect EBER RNA in oral hairy leukoplakia, a pathologic process previously characterized as associated with lytic EBV infection. The sensitivity, specificity and rapidity of this technique make it ideal for the diagnostic detection of EBV in latently infected clinical specimens.

New vancomycin disk diffusion breakpoints for enterococci. The National Committee for Clinical Laboratory Standards Working Group on Enterococci.

Since 1988, when the first vancomycin-resistant enterococcus was described, several descriptions of failures of disk diffusion breakpoints to detect low-level vancomycin resistance (MICs, 8 to 32 micrograms/ml) have been published. A four-laboratory collaborative study was undertaken to establish more accurate breakpoints for the disk test. Mueller-Hinton agar was used to perform dilution testing (in three laboratories) and disk diffusion testing (in all laboratories). Results were determined at 18, 24, and 48 h, and zones of inhibition were read using both transmitted and reflected light. One hundred organisms (35 Enterococcus faecalis, 55 E. faecium, and 10 E. gallinarum or E. casseliflavus isolates) were selected to represent vancomycin-susceptible and -resistant phenotypes. Interlaboratory agreement of agar dilution MICs was better at 24 h (91 to 94% within +/- 1 dilution) than at 18 h (76% within +/- 1 dilution). Therefore, 24-h agar dilution MIC results were used as the reference. For disk diffusion, it was critical to note the presence of a haze or colonies inside the zone when interpreting the test, since this correlated better with the results of the agar dilution test. The presence of a haze or inner colonies was best detected by reading the zones with transmitted light and incubating the plates for a full 24 h. When plotted against 24-h agar dilution MICs, breakpoints of /= 17 mm (susceptible) resulted in 58 minor errors (14.5% of total values) and 5 very major errors (2.2% of resistant values or 1.3% of total values). No major errors were seen. Results of repeat testing using a common lot of Mueller-Hinton agar showed 52 minor errors (13.3%) and 4 major errors (4.2% of susceptible values of 1.0% pf total values) but no very major errors. It is recommended that any haze or colonies within the zone be taken into account when determining zones of inhibition and that an MIC test be performed for strains with intermediate zones if vancomycin is being considered for treatment.

In situ localization of Epstein-Barr virus in thymic carcinoma.

Epstein-Barr virus (EBV) genome is associated with a variety of lymphoid and epithelial malignancies. EBV DNA has been detected in some cases of thymic carcinoma, but the cellular locus of the virus has never been defined. Detection of EBV has also been reported in normal thymus, thymic lymphoid hyperplasia, and thymoma by some investigators but not by others. In order to better define the association of the virus with benign and malignant thymic tissues and to characterize its cellular locus, we applied a recently developed in situ hybridization technique using a very abundant EBV transcript (EBER1) as target to a variety of thymic tissues. We detected expression of this transcript only in the malignant epithelial cells in one case of thymic lymphoepithelioma-like carcinoma. EBV expression was not detected in six other cases of thymic carcinoma, nor in tissue from 16 normal thymuses, 14 thymomas, and 10 thymic lymphoid hyperplasias.

Detection of herpes simplex virus by a nonradiometric spin-amplified in situ hybridization assay.

An in situ hybridization kit (Diagnostic Hybrids, Inc., Athens, Ohio) was evaluated for use in the detection and identification of herpes simplex virus (HSV) from clinical specimens. For in situ hybridization, a 10-min spin amplification onto monolayers of African green monkey kidney cells (CV-1) in 24-well polystyrene dishes, 24-h culture amplification, and hybridization with an alkaline phosphatase-labeled DNA probe were used. A total of 648 specimens were tested, including 275 specimens from patients with symptomatic diseases sent specifically for HSV detection and 373 specimens from asymptomatic immunocompromised patients sent for detection of HSV shedding. Overall, the sensitivity of the hybridization assay was 97.8% (131 of 134 specimens), with 105 of 105 (100%) specimens from symptomatic patients and 26 of 29 (89.9%) specimens from asymptomatic patients being detected. The three specimens that were false negative by in situ hybridization had low virus titers, as determined by tissue culture. The specificity was 99.6% (512 of 514 specimens). The rapid, accurate results suggest that the in situ hybridization kit may be used as an alternative to conventional tissue culture for the detection of HSV.

Prevalence of infection with human immunodeficiency virus in elective surgery patients.

An anonymous survey of elective surgery patients was performed to assess prevalence of antibody to human immunodeficiency virus (HIV) in a large urban hospital. Of 4087 patients evaluated, 18 (0.4%) were found to be infected with HIV as confirmed by a positive Western blot antibody test. Assessment of risk factors demonstrated that patients with a history of a blood transfusion did not differ in demographics or rate of infection from the population as a whole. Of the 18 HIV infected patients, 13 gave an admission history of one or more risk factors, including 10 with a history of a prior positive test. Only five, or 0.12% of the patients, provided no history of a risk factor or a history of transfusion only. The authors conclude that the prevalence of HIV infection among elective surgery patients is low, and that there would not be any substantial benefit from screening such patients for antibody against HIV.