Evaluation of CMV viral load using TaqMan CMV quantitative PCR and comparison with CMV antigenemia in heart and lung transplant recipients.

BACKGROUND: Quantitative assessment of cytomegalovirus (CMV) infection using the antigenemia test has been used to monitor CMV infection in heart and lung transplant patients enabling a preemptive treatment strategy. However, the method is labour intensive, samples have to be processed within a few hours and requires skilled interpretation. A comparative prospective evaluation of a real-time TaqMan CMV quantitative PCR (QPCR) with the CMV antigenemia was undertaken. METHODS: A real-time quantitative TaqMan CMV PCR from EDTA bloods was developed. In this study 25 heart transplant and single-lung transplant patients were monitored posttransplantation by antigenemia and TaqMan CMV QPCR. CMV DNA extracted from EDTA blood was amplified by TaqMan QPCR using primers and probe designed from the CMV glycoprotein B (gB) gene. Quantification of the genome copies is extrapolated from a standard curve generated from amplification of quantified standards. RESULTS: Antigenaemia levels and TaqMan CMV QPCR genome copies showed a linear correlation between the two assays (R=0.843, P=0.001). A clinically significant threshold of 50 CMV pp65 antigen positive polymorphonuclear leucocytes (PMNLs) per 200 000 cells previously reported was used to extrapolate an equivalent value of 40 000 (log 4.6) genome copies per ml of blood for the TaqMan CMV QPCR. CONCLUSIONS: The TaqMan system enables a rapid high-throughput of samples. The TaqMan CMV QPCR can be used as an accurate and robust alternative to the antigenemia test to predict CMV disease and to monitor effectiveness of treatment.     

荧光定量PCR技术在疟疾诊断中的应用

目的 探讨荧光定量PCR技术在疟疾诊断中的临床应用价值.方法 收集本院2010年1月至2012年3月疑似疟疾患者的血样共67份和健康人血样20份,采用荧光定量PCR法进行疟原虫检测,并与镜检法和胶体金免疫层析法的敏感性和特异性进行对比分析.对临床确诊的20例疟疾患者在抗疟药治疗前后的虫体密度与3种方法检测的疟原虫结果进行对比分析.结果 67份患者血样用荧光定量PCR法、镜检法和胶体金免疫层析法检测的疟原虫阳性率,分别为62.7%、56.7%和52.2%,差异无统计学意义(P= 0.471).3种方法检测20份健康人血样结果均为疟原虫阴性,特异性为100%.荧光定量PCR检测结果与镜检虫体密度呈直线相关(r= 0.958,P= 0.042).原虫血症较高水平时3种方法的检测结果差异无统计学意义(P> 0.05);在药物治疗后原虫血症较低水平时,虫体密度< 50/μl时,镜检法与胶体金免疫层析法检测结果均为阴性,而荧光定量PCR法检测仍为阳性,总疟原虫拷贝数均值为2.92 × 102 拷贝/ml,提示荧光定量PCR检测方法的敏感性高于镜检法和和胶体金免疫层析法.结论 荧光定量PCR方法检测疟原虫快速敏感,且特异性强,可作为镜检法的补充.     

Quantification of cytomegalovirus (CMV) viral load by the hybrid capture assay allows for early detection of CMV disease in lung transplant recipients.

BACKGROUND: We prospectively compared the hybrid capture system (HCS) assay with conventional cell culture and shell vial assay for the detection of cytomegalovirus (CMV) infection and disease in the lung transplant population. METHODS: Between January 1999 and February 2000, 34 lung transplant patients at Loyola University Medical Center, who were considered to be at risk for CMV disease, underwent surveillance testing for CMV cell culture, shell vial assay and HCS assay according to a pre-determined schedule. In addition, bronchoscopy with bronchoalveolar lavage (BAL) and transbronchial biopsy were performed at regular intervals and for clinical indications. All BAL samples were sent for CMV cultures and biopsy specimens were analyzed for histopathologic evidence of CMV by immunoperoxidase staining using antibody to early immediate nuclear antigen. RESULTS: Ten patients developed CMV disease/syndrome during the course of the study. The sensitivity, specificity, positive predictive value and negative predictive value were >90% for the HCS assay. The sensitivity of the HCS assay (90%) was statistically significantly higher than the sensitivity of either the SV assay (40%) or the cell culture (50%). In addition, the HCS assay was able to detect CMV 50 +/- 67 days prior to clinical evidence of CMV disease and an average of 36 days prior to the other detection techniques. CONCLUSION: The HCS assay is a sensitive diagnostic technique able to reliably detect CMV disease earlier than other diagnostic methods in the lung transplant population. Future studies may be able to evaluate whether pre-emptive anti-viral therapy targeted to specific viral loads using the HCS assay will be beneficial in preventing morbidity associated with CMV disease.     

Utility of Gram''s stain and efficacy of quantitative cultures for posttraumatic pneumonia: a prospective study.

OBJECTIVE: This prospective trial examined the efficacy of using bronchoalveolar lavage (BAL) for the diagnosis of pneumonia (PN) and the utility of Gram's stain (GS) for dictating empiric therapy. SUMMARY BACKGROUND DATA: Posttraumatic nosocomial PN remains a significant cause of morbidity and mortality. However, its diagnosis is elusive, especially in multiply injured patients. The systemic inflammatory response syndrome of fever, leukocytosis, and a hyperdynamic state is common in trauma patients, especially patients with pulmonary contusion. Bronchoscopy with BAL with quantitative cultures of the lavage effluent may distinguish between PN and systemic inflammatory response syndrome, and GS of the lavage effluent may guide empiric therapy before quantitative culture results. METHODS: Mechanically ventilated trauma patients with a clinical diagnosis of PN (fever, leukocytosis, purulent sputum, and new or changing infiltrate on chest radiograph) underwent bronchoscopy with BAL. Effluent was sent for GS and quantitative cultures. The diagnostic threshold for PN was > or =10(5) colony-forming units (CFU)/mL, and antibiotics were continued. Antibiotics were stopped for < 10(5) CFU/mL and the diagnosis of systemic inflammatory response syndrome was made. Causative organisms for PN were compared to GS. RESULTS: Over a 2-year period, 232 patients underwent 443 bronchoscopies with BAL (71% men, 29% women; mean age, 41). The mean injury severity score was 30. Sixty percent of the patients had pulmonary contusion, and 59% were cigarette smokers. The overall incidence of PN was 39% and was no different regardless of the number of BALs a patient had. The false-negative rate of BAL was 7%. GS identified gram-positive organisms in 80% of patients with gram-positive PN and 40% of patients with gram-negative PN. GS identified gram-negative organisms in 52% of patients with gram-positive PN and 77% with gram-negative PN. The duration of the intensive care unit stay relative to the timing of BAL was beneficial for guiding empiric therapy. BAL in week 1 primarily identified Haemophilus influenzae and gram-positive organisms; Acinetobacter sp. and Pseudomonas sp. were more common after week 1. CONCLUSIONS: Bronchoscopy with BAL is an effective method to diagnose PN and avoids prolonged, unnecessary antibiotic therapy. Empiric therapy should be adjusted to the duration of the intensive care unit stay because the causative bacteria flora changes over time. GS of BAL effluent correlates poorly with quantitative cultures and is not reliable for dictating empiric therapy.     

Video-assisted thoracoscopic lung biopsy in the diagnosis of diffuse interstitial lung disease. A prospective study

AIM: Video-assisted thoracoscopic lung biopsy is commonly performed for diagnosis of diffuse interstitial lung disease. This study reports our experience with this technique in terms of diagnostic accuracy, complications and appropriate site for biopsy. METHODS: A prospective non-randomized study. SETTING: Thoracic Surgery Department, Chest Diseases Hospital, Kuwait. PATIENTS AND INTERVENTION: 79 consecutive patients undergoing thoracoscopic lung biopsy for diffuse interstitial lung disease from January 1995 to December 2001. Patients were divided into 2 groups; 34 patients with single biopsy from lingula or right middle lobe (RML) and 45 with 2 biopsies from other sites. Measures: comparison of operative and postoperative data, diagnostic accuracy, and complications between the two groups. RESULTS: The mean age of the patients was 38.9 years (range 15-75 years). There was no difference in the groups in operative time, 24 hours postoperative pleural drainage, chest tube drainage days, and hospital stay. Histologic diagnosis was achieved in 76/79 patients (96%). The diagnostic yield of each group was comparable (32/34 from lingula/RML; 44/45 other sites), (p=0.3). Postoperative complications occurred in 4/34 of patients with single biopsy from lingula or RML and 4/45 patients with 2 biopsies from other sites (p=0.6). There was 1 death in this series. CONCLUSIONS: Video-assisted thoracoscopic lung biopsy is an effective procedure. Single lung biopsy from lingula or RML compared to other anatomic sites has an equivalent diagnostic yield.     

A prospective study of protected bronchoalveolar lavage in the diagnosis of nosocomial pneumonia

The value of a new technique of protected bronchoalveolar lavage not requiring bronchoscopy was prospectively evaluated for the diagnosis of nosocomial pneumonia in two groups of critically ill patients. The control group was comprised of 29 patients free of any pulmonary disease whose lungs were ventilated for a mean time of 14 +/- 9 days (mean +/- SD). The pneumonia group was comprised of 30 patients with histologically proven nosocomial pneumonia whose lungs were ventilated for a mean time of 11 +/- 8 days. All patients of the pneumonia group died, and postmortem lung biopsies were taken for bacteriologic and pathologic examination. Twice a week in the control group or within the day preceding death in the pneumonia group, distal bronchial samples were obtained by a technique of protected bronchoalveolar lavage performed at the bedside. Fifty-one distal bronchial samples were bacteriologically analyzed in the control group and 30 in the pneumonia group. The sensitivity of a positive protected bronchoalveolar lavage for diagnosing nosocomial pneumonia was 80%, whereas the specificity was 66%. In 73% of the patients of the pneumonia group, the microorganisms isolated in the protected bronchoalveolar lavage and in the lung culture were partially (16%) or completely in agreement (57%). Among the 43 microorganisms isolated in the lung cultures, 74% were recovered by the protected bronchoalveolar lavage technique. This study shows that the protected bronchoalveolar lavage is an accurate technique for the diagnosis of nosocomial pneumonia. Because the technique is simple, noninvasive, easily repeatable at the bedside, and enables gram staining, it represents an attractive alternative to the fiberoptic bronchoscopy technique using a plugged double-sheathed brush.     

动态检测血浆CMV DNA载量在预测肾移植术后并发巨细胞病毒肺炎中的意义

目的探讨肾移植受者巨细胞病毒(CMV)肺炎与病毒载量的关系,寻找预测CMV肺炎的病毒载量阈值。方法应用RT-PCR方法定期随访检测56例肾移植受者血浆中CMVDNA载量。比较CMV肺炎组和非肺炎组术后各时点的载量平均值,筛选预测CMV肺炎的最合适阈值。结果56例患者发生CMV肺炎8例(14.3%)。移植后4周2组CMVDNA载量均在0值附近,第5周肺炎组载量开始上升,第8周达到峰值,然后迅速下降;而非肺炎组观察期间载量均在低水平波动。5~11周期间肺炎组载量平均值高于非肺炎组,其中5、7、9周载量值差异有统计学意义(P<0.05)。以术后5~15周内连续3次CMVDNA载量>1×104拷贝/ml作为预测CMV肺炎指标,灵敏度、特异度、阳性预测值、阴性预测值分别为62.5%、93.8%、62.5%、93.8%,Kappa值0.5625(95%CI=0.2503~0.8747),预警时间2~10周。结论肾移植后CMV肺炎发病前血浆CMVDNA载量值呈持续高水平状态,5~15周内连续3次CMVDNA载量>1×104拷贝/ml是预测CMV肺炎的良好指标。     

Role of CMV pneumonia in the development of obliterative bronchiolitis in heart-lung and double-lung transplant recipients

Obliterative bronchiolitis (OB) is the main cause of late mortality after lung transplantation. Cytomegalovirus infection has been associated with late graft failure. The aim of this study was to determine whether the development of OB was related to CMV pretransplant serological status and to CMV infections. The study group comprised 36 lung transplant recipients (27 HLT and 9 DLT) who survived more than 4 months, of whom 47% developed OB (defined by the persistence of an unexplained obstructive disease: FEV1/VC < 0,7). OB occurred more frequently: (1) in seronegative recipients with seropositive donors (8/9) than in seropositive recipients (7/19) or seronegative well-matched recipients (2/8); and (2) in patients who experienced CMV pneumonia (11/16) and CMV recurrence (11/16). Since matching seronegative recipients is the best way to prevent CMV infection, we believe that seronegative grafts must be reserved for seronegative recipients.     

A prospective study of quantitative ultrasound in children and adolescents.

The accrual of optimal bone mass during childhood and adolescence is essential for the formation of a skeleton that will meet structural needs throughout life. Assessing bone health of children is becoming increasingly important in order to identify those who require interventions, and quantitative ultrasound (QUS) has appeal for these assessments. The purpose of this prospective study was to characterize changes in QUS values in 328 healthy children and adolescents over a 3-yr period. Measurements of QUS, height, weight, nutrient intake, fracture history, and Tanner stage were made at baseline and 3 yr later. Both females and males experienced significant increases in QUS values during the study. The rate of change of QUS peaked at an earlier age in females than in males, and maximum accumulation rates in both genders occurred at ages at which highest accumulation rates are seen with densitometry. Females exhibited higher QUS values than males during puberty, also similar to results for dual X-ray absorptiometry (DXA). This is the first report of prospective data of QUS in children and adolescents. Our findings that QUS values change during childhood and adolescence in a manner similar to DXA values, the "gold standard," provide support for the validity of using QUS to assess bone health in children and adolescents.     

A prospective study of anaemia and long-term outcomes in kidney transplant recipients.

BACKGROUND: Anaemia is prevalent in kidney transplant recipients (KTR), and only few KTR with anaemia receive treatment with erythropoietin. Some have claimed that this undertreatment might contribute to suboptimal outcomes such as mortality and cardiovascular events in these patients. However, no evidence is currently available that anaemia is actually associated with such risks in KTR. METHODS: We merged two cohorts of KTR to study the associations between anaemia and two outcomes: all-cause mortality and kidney allograft loss. Detailed information on the demographic and clinical characteristics of these 825 patients was available at baseline. As recommended by the American Society of Transplantation, anaemia was considered present if the haemoglobin concentration was < or =13 g/dl in men or < or =12 g/dl in women. Patients were followed using the Austrian Dialysis and Transplant Registry. RESULTS: After 8.2 years of follow-up, 251 patients died and 401 allografts were lost. In multivariate analyses, anaemia was not associated with all-cause mortality (HR: 1.08; 95% CI: 0.80-1.45), but it was associated with 25% greater risk of allograft loss (HR = 1.25; 95% CI: 1.02-1.59). This association was even more pronounced in death-censored analyses. Analyses using haemoglobin as a continuous variable or in categories also found no association with mortality. CONCLUSIONS: Anaemia may not be associated with mortality in KTR. In light of the recent findings of increased mortality in chronic kidney disease patients with higher haemoglobin treatment target, further evidence is needed to guide clinicians in the treatment of anaemia in these patients.     

T淋巴细胞亚群检测在肾移植排斥反应与巨细胞病毒感染鉴别诊断中的应用

目的:探讨外周血淋巴细胞亚群的检测在人类同种异体肾移植术后急性排斥反应与巨细胞病毒(CMV)感染的诊断与鉴别诊断中的价值。方法:采用三色流式细胞技术对47例肾移植术后肾功能正常、16例急性排斥反应、11例CMV感染三组患者外周血淋巴细胞中CD3+、CD3+CD4+、CD3+CD8+细胞的百分比进行检测,并计算出CD3+CD4+细胞与CD3+CD8+的比值,分析不同T淋巴细胞亚群在三组患者中的差别,并行t检验。结果:肾功能正常组、急性排斥反应组与CMV感染组外周血淋巴细胞中CD3+细胞的百分比分别为(70.33±10.96)%、(74.46±8.78)%和(74.06±12.94)%,差异无统计学意义;CD3+CD4+细胞的百分比分别为(40.85±9.58)%、(50.85±8.43)%和(28.62±9.40)%,急性排斥反应组及CMV感染组与肾功能正常组相比,差异均有统计学意义(t=3.871;P<0.01。CD3+CD8+淋巴细胞的百分比分别为(27.08±9.40)%、(20.15±5.47)%和(46.32±15.51)%,急性排斥反应组及CMV感染组与肾功能正常组相比,差异均有统计学意义(t=2.787,P<0.01;t=5.346,P<0.01);CD3+CD4+/CD3+CD8+分别为1.78±0.98、2.88±0.76和0.69±0.31,急性排斥反应组及CMV感染组与肾功能正常组相比,差异均有统计学意义(均P<0.01)。结论:检测外周血淋巴细胞亚群的变化,特别是CD3+CD4+/CD3+CD8+比值,对肾移植术后急性排斥反应及CMV感染具有鉴别诊断价值。     

A retrospective assessment of safety, efficacy, and pharmacoeconomics of generic azathioprine in heart-transplant recipients.

Although a generic formulation of azathioprine (AZA) has been available since 1996, safety, efficacy and pharmacoeconomic implications following conversion from Imuran (AZA) to generic AZA in heart-transplant patients remains to be determined. A retrospective, safety and efficacy assessment, in addition to a cost comparison, was performed in 30 heart-transplant patients who had been switched from Imuran to generic AZA. In heart-transplant patients converted from Imuran to generic AZA, no compromise in safety and efficacy, as measured by white blood cell (WBC) count, infections, rejections, malignancies, and hospitalizations was observed. Generic substitution of Imuran results in an annual cost savings of $318 per patient.     

A prospective study of the physician effect on blood pressure in renal-transplant recipients.

BACKGROUND: Physician presence results in elevated blood pressure (BP) in the general population. The determinants of this phenomenon in renal-transplant recipients are not known. METHODS: We prospectively evaluated BP changes with physician presence in 231 stable adults with graft survival > or =1 year. A nurse measured timed sitting BP by Korotkoff phase I and V sounds before physician entry, during physician presence and upon exit. The haemoglobin, creatinine, weight, immunosuppressive drug dosage and/or level, and anti-hypertensive medication profile were recorded. Paired Student's t-test with Bonferroni correction and multiple linear regression analysis were used to examine BP changes. Characteristics of patients with change in mean arterial BP > or =+10 mmHg (n=55, "high") were compared with those with change < or =+5 mmHg (n=132, "low") by chi-square or Wilcoxon rank sum test as appropriate. A group of 100 recipients served as controls in whom BP was measured thrice without physician presence on any occasion. A multivariate analysis was performed for the combined groups controlling for physician entry as a predictor variable. RESULTS: In the study group, systolic BP (mean+/-SE) rose by 4.2+/-0.8, diastolic BP by 3.5+/-0.5 and mean arterial BP by 3.5+/-0.5 mmHg in physician presence. The BP returned to baseline with exit (P<0.001 for each). Higher haemoglobin and creatinine demonstrated a trend towards influencing the physician-induced rise in diastolic BP. The "high" sub-group demonstrated a shorter time to the second BP measurement in physician presence (P=0.03) and a trend towards morning measurements (P=0.08). In the control group, systolic BP declined by 3.2+/-0.9 mmHg from the first to the second measurement, with a further decline of 2.4+/-0.6 mmHg from the second to third measurements. Diastolic BP did not change. In the combined multivariate analysis, physician entry was an independent predictor of BP change (P=0.0001). CONCLUSIONS: Renal-transplant recipients demonstrate a significant physician effect on BP despite adequate experience with post-transplant clinic visits and BP-altering medication. This population deserves further evaluation through ambulatory BP monitoring.     

巨细胞病毒抗原检测对巨细胞病毒病预防的意义

目的：提高对肾移植术后巨细胞病毒（CMV）病的防治水平,方法：应用免疫组织化学方法（LSAB法）对1997年度肾移植患者进行CMV抗原检测,对检查阳性的患者采用丙氧鸟苷预防性治疗,并与1996年度未预防治疗组进行对照.结果：1997年预防性治疗后,CMV病发病率为3．1％（5／160）,病死率为20．0（1／5）,而1996年未预防治疗组,CMV病发病率为11．7％（16／136）,病死率为43．8％（7／16）,结论：对肾移植术后CMV抗原检测阳性患者预防性应用丙氧鸟式能显著降低CMV病的发生率和病死率。     

Impact of prophylaxis with cytogam alone on the incidence of CMV viremia in CMV-seropositive lung transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) infection remains a serious problem after lung transplantation. The purpose of this study was to evaluate the efficacy of CytoGam, a CMV hyperimmune globulin (CMV-IGIV), as CMV prophylaxis after lung transplantation. METHODS: This prospective, randomized, open-label study compared prophylaxis with CMV-IGIV and no prophylaxis in 44 CMV-seropositive lung transplant recipients. The primary end-point was development of CMV viremia during the first year after transplantation. RESULTS: Cytomegalovirus viremia was detected in 13 of 22 recipients without prophylaxis and in 16 of 22 recipients with CMV-IGIV prophylaxis (p = 0.19). Cytomegalovirus pneumonitis developed in 8 controls vs in 11 CMV-IGIV recipients (p = 0.54). We found no significant difference between the groups in the incidence of positive shell vial assays (6.8% +/- 6.5% without vs 11.2% +/- 10.1% with prophylaxis, p = 0.09) or in the attack rate of CMV pneumonitis (0.41 +/- 0.59 episodes/patient without vs 0.86 +/- 0.99 episodes/patient with prophylaxis, p = 0.07). Similarly, no difference was apparent in the time to onset of CMV viremia, to detection of CMV DNA in peripheral blood leukocytes by polymerase chain reaction, or to development of CMV pneumonitis. The incidence of acute rejection and bronchiolitis obliterans syndrome and the survival rate during the first post-transplant year did not differ between the groups. CONCLUSIONS: Prophylaxis with CMV-IGIV alone did not decrease CMV viremia or pneumonitis, did not decrease the incidence of acute rejection or bronchiolitis obliterans syndrome, and did not affect 1-year survival of CMV-seropositive lung transplant recipients at our center.     

The enzyme-linked immunosorbent assay (ELISA) in the laboratory diagnosis of gonorrhoea. A comparative evaluation

An enzyme-linked immunosorbent assay (ELISA) was used to detect levels of antibodies to Neisseria gonorrhoeae in 100 male patients with suspected gonorrhoea attending a clinic for sexually transmitted diseases. Although significantly higher antibody levels were detectable in patients with clinically diagnosed gonorrhoea confirmed by laboratory tests, positive results were also obtained in 26% of the control subjects, who were presumed to be healthy. Serum anticomplement activity was present in 8% of the subjects with false-positive results, while tests for rheumatoid factor were positive in 14%. However, these problems could be eliminated to a certain extent by including a negative control or background well for each serum sample. Because the ELISA was found to be of high sensitivity and moderate specificity it is concluded that it is a useful serodiagnostic test for gonorrhoea.     

Viral infections in renal transplant donors and their recipients: a prospective study.

The majority of renal allograft recipients develop viral infections, usually with cytomegalovirus (CMV). Their source if virus has not been defined clearly; one possibility if the transplanted kidney itself. To explore this, prospective viral studies were performed on 28 living related donor-recipient pairs. Donors did not have clinical illnesses and viruses were not recovered from throat, urine, or renal tissue, but five (18%) had fourfold rises in antibody titers to herpes group viruses. During the 6 months after transplantation, 24 recipients (86%) had viral infections, 18 of which were associated with CMV. There was no correlation between specific titer rises in the donors and infections in the recipients. Recipients with dual viral infections had more severe clinical courses than those with single infections or with no infection. Recipients with complement-fixing (CF) antibodies to CMV pretransplant had a higher incidence of CMV infections than recipients without pretransplant antibody. Three of seven recipients who lacked CF antibody to CMV and whose donors were seropositive developed clinical illnesses associated with CMV. Latent virus might have been transmitted with the transplanted kidney in these instances, but since lack of CF antibody does not rule out previous CMV infection, the CMV could have been of recipient origin. We conclude that the donor organ is a source of virus for few renal transplant recipients.     

Early and rapid diagnosis of CMV infection by nonradioactive in situ hybridization in pediatric kidney transplant recipients.

A nonradioactive in situ hybridization technique was utilized for the rapid and early diagnosis of cytomegalovirus (CMV) infection in children undergoing kidney transplantation. The cellular samples were obtained directly from the organs thought to be affected on the basis of clinical findings: bronchoalveolar lavage during interstitial pneumonia (7 samples from 6 cases); fine-needle aspiration biopsy (FNAB) of the liver during acute hepatitis (1 case); kidney FNAB and peripheral blood where there was a greater than or equal to 25% creatinine rise with or without fever (26 episodes). Standard virus isolation procedures and an immunofluorescent technique on short-term cultures of human fibroblast cells were performed as a control. 6/23 children followed had a symptomatic CMV infection (4 had interstitial pneumonia; 1 had acute hepatitis, and there was 1 case of creatinine rise with fever). In all cases, the diagnosis was provided by in situ hybridization in less than 24 h. These results were confirmed 48 h later by immunofluorescence and after 5-25 days by standard viral cultures. In situ hybridization with a biotinylated probe proved to be a rapid and sensitive method for diagnosis of CMV disease, when performed on specimens obtained from the involved organs at an early stage of the infection. This diagnostic approach allowed a specific antiviral therapy to be undertaken promptly.