Imaging mass spectrometry: a new way to distinguish dermal contact from administration of cocaine, using a single fingerprint.

Here we show a new and significant application area for mass spectrometry imaging. The potential for fingerprints to reveal drug use has been widely reported, with potential applications in forensics and workplace drug testing. However, one unsolved issue is the inability to distinguish between drug administration and contamination by contact. Previous work using bulk mass spectrometry analysis has shown that this distinction can only be definitively made if the hands are washed prior to sample collection. Here, we illustrate how three mass spectrometry imaging approaches, desorption electrospray ionisation (DESI), matrix assisted laser desorption ionisation (MALDI) and time of flight secondary ion mass spectrometry (ToF-SIMS) can be used to visualise fingerprints at different pixel sizes, ranging from the whole fingerprint down to the pore structure. We show how each of these magnification scales can be used to distinguish between cocaine use and contact. We also demonstrate the first application of water cluster SIMS to a fingerprint sample, which was the sole method tested here that was capable of detecting excreted drug metabolites in fingerprints, while providing spatial resolution sufficient to resolve individual pore structure. We show that after administration of cocaine, lipids and salts in the fingerprint ridges spatially correlate with the cocaine metabolite, benzoylecgonine. In contrast after contact, we have observed that cocaine and its metabolite show a poor spatial correlation with the flow of the ridges.

Borderline anaemia and postoperative outcome in women undergoing major abdominal surgery: a retrospective cohort study.

Pre-operative anaemia is typically diagnosed with a haemoglobin concentration < 120 g.l-1 for women and < 130 g.l-1 for men on the basis of limited evidence. This retrospective cohort study stratified women undergoing elective, major abdominal surgery based on pre-operative haemoglobin concentration: anaemic (< 120 g.l-1 ); borderline anaemic (120-129 g.l-1 ); and non-anaemic (> 130 g.l-1 ). Data from 1554 women were analysed. Women with borderline anaemia had a greater incidence of postoperative complications (55 (16%) vs. 110 (11%); p = 0.026), longer duration of hospital stay (median (IQR [range]) 3 (1-6 [0-69]) days vs. 2 (1-5 [0-80]) days; p = 0.017) and fewer days alive and out of hospital at postoperative day 30 (median (IQR [range]) 27 (23-29 [0-30]) vs. 28 (25-29 [0-30]) days; p = 0.017) compared with non-anaemic women. However, after matched cohort analysis, these outcome differences no longer remained statistically significant. After multivariable adjustment for procedure, Charlson comorbidity index and patient age, a negative relationship between logarithmic pre-operative haemoglobin concentration and duration of stay was found (parameter estimate (standard error) -0.006 (0.003) vs. 0.003 (0.003) for a haemoglobin concentration < 130 g.l-1 vs. > 130 g.l-1 , respectively; p = 0.03); the difference in duration of stay was approximately 50% greater for women with a haemoglobin concentration of 120 g.l-1 compared with those with a haemoglobin concentration of 130 g.l-1 . Although the contribution of borderline anaemia to the incidence of postoperative complications is uncertain, the current diagnostic criteria should be re-assessed.

Diabetes is a major risk factor for mortality after lung transplantation.

Survival following lung transplant (LTx) remains significantly lower than after other solid organ transplants. Diabetes mellitus (DM) is a mortality risk factor not comprehensively studied in LTx recipients. Notably, neither the relation of time of DM onset to survival nor the actual causes of DM-associated excess mortality have been described. We determined DM status, DM diagnosis date and all-cause mortality in 386 consecutive adults who underwent LTx at our institution from January 1, 2001 to July 31, 2010. The relationship of DM to survival both as a categorical and time-dependent variable was studied. Fifty-three percent of patients had DM. Overall median survival was 5.2 (95% CI 3.8-6.6) years. At study end, 52% of patients had died, of whom 64% had DM. Estimated median survival was 10 years in patients without DM, 5.0 (3.3-6.8) years in patients with DM pre- and post-LTx and 4.3 (3.1-5.5) years in patients with new onset DM. As a time-dependent covariate, DM was the strongest risk factor for mortality, hazard ratio 3.96 (2.85-5.51). Bronchiolitis obliterans syndrome was the main cause of death in all patients surviving >90 days, but its incidence was not increased in patients with DM. Further studies are warranted to determine whether improved glycemic control could improve outcomes in LTx recipients.

A multifaceted pharmacist intervention to improve antihypertensive adherence: a cluster-randomized, controlled trial (HAPPy trial).

WHAT IS KNOWN AND OBJECTIVES: About half of all patients taking antihypertensives discontinue treatment by 12 months. There is potential for substantial health gains at both individual and population levels through improved treatment adherence. The objective was to evaluate a community pharmacist intervention to improve adherence with antihypertensive medicines with a view to improving blood pressure (BP) control. DESIGN: prospective, non-blinded, cluster-randomized, controlled trial. PARTICIPANTS: adults with primary hypertension who obtained antihypertensives in the previous 6 months. Patients with poor refill adherence were preferentially identified with the help of a purpose-built software application. INTERVENTION: package comprising BP monitor; training on BP self-monitoring; motivational interviewing; medication use review; prescription refill reminders. FOLLOW-UP: six months. PRIMARY OUTCOME: change in proportion self-reporting medication adherence. Secondary outcome: BP changes. RESULTS: Participants (n = 395; intervention - 207; control - 188) had a mean age of 66.7 years; 51.1% were males. The proportion of adherent participants increased in both groups but was not significantly different between groups [57·2% to 63·6% (control) vs. 60·0% to 73·5% (intervention), P = 0·23]. The mean reduction in systolic BP was significantly greater in the intervention group (10·0 mmHg vs. 4·6 mmHg; P = 0·05). The proportion of patients who were non-adherent at baseline and adherent at 6 months was 22·6% (95%CI 5·1-40·0%) higher in the intervention group (61·8% vs. 39·2%, P = 0·007). Among participants with baseline BP above target, reduction of systolic BP was significantly greater in the intervention group [by 7·2 mmHg (95%CI 1·6-12·8 mmHg); (P = 0·01)]. Among participants non-adherent at baseline and above target BP, the proportion reporting adherence at 6 months was significantly greater in the intervention group [56·8% vs. 35·9%, P = 0·039). WHAT IS NEW AND CONCLUSION: This community pharmacist intervention resulted in improved adherence to antihypertensive medication and reduced systolic BP.

Measurement of isoniazid in tuberculosis patients using finger sweat with creatinine normalisation: A controlled administration study.

BACKGROUND: Insufficient exposure and poor compliance with anti-tuberculosis (TB) medications are risk factors for treatment failure and the development of drug resistance. Measurement of drugs in biological samples, such as blood and saliva, can be used to assess adherence and make dose adjustments by therapeutic drug monitoring (TDM). Finger sweat testing is a convenient and non-invasive method to monitor patients. OBJECTIVES: To assess the feasibility of finger sweat testing for medication adherence and as a semi-quantitative tool for TDM analysis. METHODS: Ten patients provided finger sweat, blood and saliva samples following a controlled dose of isoniazid. Samples were analysed by liquid chromatography-mass spectrometry. RESULTS: Isoniazid can be detected in finger sweat 1-6 h following administration at typically prescribed dosages. The normalisation of isoniazid to creatinine increases the correlation between finger sweat and serum isoniazid concentration and provides a means to account for inconsistent sample volumes. CONCLUSION: We describe the time-course measurement of isoniazid (or drug-to-creatinine ratio) in finger sweat compared to the pharmacokinetic profile in blood for the first time. This technique, adaptable for other drugs, could reduce the burden on clinics and improve patient experience.

Enhanced imaging of developed fingerprints using mass spectrometry imaging.

Latent fingermarks are invisible to the naked eye and normally require the application of a chemical developer followed by an optical imaging step in order to visualize the ridge detail. If the finger deposition is poor, or the fingermark is aged, it can sometimes be difficult to produce an image of sufficient quality for identification. In this work, we show for the first time how mass spectrometry imaging (in this case time-of-flight secondary ion mass spectrometry, ToF-SIMS) can be used to enhance the quality of partially recovered fingermarks. We show three examples of how chemical imaging can be used to obtain enhanced images of fingermarks deposited on aluminium foil, glass and the handle of a hand grenade compared with conventional development techniques.

Evaluation of particle-induced X-ray emission and particle-induced γ-ray emission of quartz grains for forensic trace sediment analysis.

The independent verification in a forensics context of quartz grain morphological typing by scanning electron microscopy was demonstrated using particle-induced X-ray emission (PIXE) and particle-induced γ-ray emission (PIGE). Surface texture analysis by electron microscopy and high-sensitivity trace element mapping by PIXE and PIGE are independent analytical techniques for identifying the provenance of quartz in sediment samples in forensic investigations. Trace element profiling of the quartz grain matrix separately from the quartz grain inclusions served to differentiate grains of different provenance and indeed went some way toward discriminating between different quartz grain types identified in a single sample of one known forensic provenance. These results confirm the feasibility of independently verifying the provenance of critical samples from forensic cases.

On the relevance of cocaine detection in a fingerprint.

The finding that drugs and metabolites can be detected from fingerprints is of potential relevance to forensic science and as well as toxicology and clinical testing. However, discriminating between dermal contact and ingestion of drugs has never been verified experimentally. The inability to interpret the result of finding a drug or metabolite in a fingerprint has prevented widespread adoption of fingerprints in drug testing and limits the probative value of detecting drugs in fingermarks. A commonly held belief is that the detection of metabolites of drugs of abuse in fingerprints can be used to confirm a drug has been ingested. However, we show here that cocaine and its primary metabolite, benzoylecgonine, can be detected in fingerprints of non-drug users after contact with cocaine. Additionally, cocaine was found to persist above environmental levels for up to 48 hours after contact. Therefore the detection of cocaine and benzoylecgonine (BZE) in fingermarks can be forensically significant, but do not demonstrate that a person has ingested the substance. In contrast, the data here shows that a drug test from a fingerprint (where hands can be washed prior to donating a sample) CAN distinguish between contact and ingestion of cocaine. If hands were washed prior to giving a fingerprint, BZE was detected only after the administration of cocaine. Therefore BZE can be used to distinguish cocaine contact from cocaine ingestion, provided donors wash their hands prior to sampling. A test based on the detection of BZE in at least one of two donated fingerprint samples has accuracy 95%, sensitivity 90% and specificity of 100% (n = 86).

Focal cerebral oxygenation and neurological outcome with or without brain tissue oxygen-guided therapy in patients with traumatic brain injury.

BACKGROUND: In patients with severe traumatic brain injury (TBI), the depth and duration of cerebral hypoxia are independent predictors of outcome. This study aimed to evaluate the efficacy of brain oxygen-guided therapy in improving cerebral oxygenation and neurological outcome in severe TBI patients. METHODS: Thirty TBI patients had brain oxygen monitors placed contralateral to the side of mass lesions, or to the non-dominant side if injury was diffuse. The first 10 patients (Group 1, observational) had brain tissue oxygen (PbrO2) monitored, but not treated. The next 20 patients (Group 2, interventional) were treated according to brain tissue oxygen-guided algorithms aiming to improve cerebral oxygen availability. The 6-month neurological outcome of Group 2 patients was compared with that of Group 1 patients and with contemporary control patients (Group 3) treated without the use of brain oxygen monitoring. FINDINGS: The mean duration of brain hypoxic episodes (PbrO2 <15 mmHg) was 106 minutes in Group 1, and 34 minutes in Group 2 (p=0.01). Brain tissue oxygen was <15 mmHg for 10% of monitoring time in Group 1 and 2.8% in Group 2 (p=0.12). The peak incidence of cerebral hypoxic events in both groups occurred during post-injury day 5. The mean Injury Severity Score (ISS) of patients experiencing cerebral hypoxia was higher than that of patients without cerebral hypoxic episodes (33.7 vs 24.2, p=0.04). There was no statistically significant difference in neurological outcome between those patients treated with and those without brain oxygen-guided therapy. CONCLUSIONS: In TBI patients, brain tissue oxygen-guided therapy is associated with decreased duration of episodes of cerebral hypoxia. Larger studies are indicated to determine the effects of this therapy on neurological outcome.

Exploring a route to a selective and sensitive portable system for explosive detection- swab spray ionisation coupled to of high-field assisted waveform ion mobility spectrometry (FAIMS).

Paper spray mass spectrometry is a rapid and sensitive tool for explosives detection but has so far only been demonstrated using high resolution mass spectrometry, which bears too high a cost for many practical applications. Here we explore the potential for paper spray to be implemented in field applications with portable mass spectrometry. This involved (a) replacing the paper substrate with a swabbing material (which we call "swab spray") for compatibility with standard collection materials; (b) collection of explosives from surfaces; (c) an exploration of interferences within a ±â€¯0.5 m/z window; and (d) demonstration of the use of high-field assisted waveform ion mobility spectrometer (FAIMS) for enhanced selectivity. We show that paper and Nomex® are viable collection materials, with Nomex providing cleaner spectra and therefore greater potential for integration with portable mass spectrometers. We show that sensitive detection using swab spray will require a mass spectrometer with a mass resolving power of 4000 or more. We show that by coupling the swab spray ionisation source with FAIMS, it is possible to reduce background interferences, thereby facilitating the use of a low resolving power (e.g. quadrupole) mass spectrometer.

A comparison of protein quantitation assays for biopharmaceutical applications.

Dye-based protein determination assays are widely used to estimate protein concentration, however various reports suggest that the response is dependent on the composition and sequence of the protein, limiting confidence in the resulting concentration estimates. In this study a diverse set of model proteins representing various sizes of protein and covalent modifications, some typical of biopharmaceuticals have been used to assess the utility of dye-based protein concentration assays. The protein concentration assays (Bicinchoninic acid (BCA), Bradford, 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA), DC, Fluorescamine and Quant-i) were compared to the 'gold standard' assay, quantitative amino acid analysis (AAA). The assays that displayed the lowest variability between proteins, BCA and DC, also generated improved estimates when BSA was used as a standard, when compared to AAA derived concentrations. Assays read out by absorbance tended to display enhanced robustness and repeatability, whereas the fluorescence based assays had wider quantitation ranges and lower limits of detection. Protein modification, in the form of glycosylation and PEGylation, and the addition of excipients, were found to affect the estimation of protein concentration for some of the assays when compared to the unmodified protein. We discuss the suitability and limitations of the selected assays for the estimation of protein concentration in biopharmaceutical applications.

The use of solid physical models for the study of macromolecular assembly.

The use of modern technology in the construction of accurate solid macromolecular models based on atomic coordinates and electron density functions has led us to re-examine the usefulness of physical models as tools for understanding molecular assembly and for designing detailed experimental and computational studies of the assembly process. Recent developments include the construction of new models, which have provided insights into the assembly of viruses and light harvesting complexes.

Split-thickness skin graft donor sites: a comparative study of two absorbent dressings.

OBJECTIVE: To identify the optimal dressing for split-thickness skin graft (SSG) donor sites. METHOD: This prospective randomised controlled trial compared two dressings - a new absorbent form of a polyurethane film dressing (Tegaderm Absorbent, 3M) and our standard alginate dressing (Kaltostat, ConvaTec) - on SSG donor sites in 40 patients. Primary outcome measures were: reduced time to full healing; reduced postoperative pain; reduced leakage rates from the dressing. Secondary outcome measures related to acceptability of the dressings to the patient. RESULTS: On removal of the dressings at the first assessment, 79% of the Tegaderm Absorbent donor sites had healed completely, compared with 16% of the Kaltostat ones (p<0.001).A significantly greater median area had healed with Tegaderm Absorbent (100%), when compared with Kaltostat (89%) (p<0.001). Mean time to complete healing was also significantly faster for Tegaderm Absorbent than Kaltostat (14 versus 21 days) (p<0.001). Significantly fewer subjects experienced postoperative pain with Tegaderm Absorbent on both day 1 (21% versus 67%, p=0.006, NNT=3) and day 2 (17% versus 75%, p<0.001, NNT=2). Leakage rates reduced by 48% with Tegaderm Absorbent, with no leakage in the smaller donor sites. Tegaderm Absorbent was significantly easier to apply than Kaltostat (89% versus 27% found it'very easy') as was ease of removal (84% versus 11% found it'very easy') (p<0.0001). Patients found Tegaderm Absorbent dressings significantly more convenient to manage and bathe with. At one month post-surgery, Vancouver scar scores showed thatTegaderm Absorbent donor sites were less red, flatter, softer and less itchy. CONCLUSION: Tegaderm Absorbent provides a significant improvement in terms of donor-site pain, healing and ease of management.

Road traffic injuries in the elderly.

OBJECTIVE: Elderly victims of motor vehicle collisions are increasing with the aging population. This study aimed to investigate the injury pattern of elderly victims involved in motor vehicle collisions. METHODS: This was a retrospective study using data from the Victorian State Trauma Outcome Registry and Monitoring Group (VSTORM) from June 2001 to July 2003, Australian Bureau of Statistics year 2001 population estimates, and Victoria Transport Accident Commission year 2001 total road death toll. Elderly victims were defined as age 65 and above. Comparison of fatality rates and general injury patterns for the elderly and young victims was undertaken. RESULTS: The total fatality rate of the elderly group was almost double that of the younger group. The elderly victims had a higher rate of chest injuries (23.42% v 18.17%; p = 0.003). The three most common chest injuries of the elderly victims were rib fractures (23.58%), flail chest (9.55%), and sternum fractures (5.97%). Elderly chest injured patients also had longer intensive care unit stay compared with the younger group (7.96 days v 5.31 days; p = 0.048). CONCLUSIONS: Elderly victims of motor vehicle collisions have a higher risk of chest injuries, especially of chest wall injuries. Age specific injury patterns are important in determining primary and secondary prevention strategies.

Investigation into the impact of couch sag on delivered dose.

The effect of couch sag on treatment delivery accuracy has been investigated by modelling the variation of delivered dose from planned dose due to the difference between the treatment and simulation couches. The couch sag of the Siemens (Concord, USA) Primus linac was determined relative to the couch sag of the Siemens (Germany) Sensation 4 CT Scanner. A phantom planning study was then undertaken to evaluate the likely clinical impact of the couch sag through an evaluation of changes in dose distribution, dose volume histograms and monitor units. The couch sag was simulated by altering the angle of the CT gantry when obtaining image studysets. A second investigation into the effects of couch sag was undertaken using an existing patient CT studyset. For this investigation, the couch sag was simulated by appropriate rotation of the gantry and collimator angles. The effect of couch sag on calculated monitor units (MU) was found to be statistically insignificant. The small monitor unit changes observed were likely to result from differences in the average linear attenuation coefficient along the beam path to the isocentre. The major differences seen however were in the regions away from the central axis. The dose volume histograms showed that both the bladder and rectum were further spared with increasing tilt angle whilst the PTV dose was unchanged. The only issue at South West Sydney Cancer Services (SWSCS) in terms of patient position variation arises from the angle induced by the couch sag (or more precisely, the difference in couch sag angle between the CT and Linac couches). Due to the relatively uniform structures (of PTV, bladder and rectum), and the proximity of these critical structures to the isocentre, this angular rotation about the isocentre did not cause any major variations to the DVH, MU and isodoses for realistic levels of couch sag (i.e. less than 20mm).

High central venous pressure is associated with prolonged mechanical ventilation and increased mortality after lung transplantation.

BACKGROUND: Poor oxygenation might occur in transplanted lungs as a result of reperfusion injury and lack of lymphatic drainage. Low central venous and pulmonary capillary wedge pressures are advocated to reduce pulmonary edema and maximize oxygenation but might adversely affect cardiac index, circulation, and renal function. METHODS: Histories, intensive care unit charts, and donor data on 118 lung transplantations performed between 1999 and 2002 were retrospectively assessed. Multiple logistic regression analysis was performed on donor, recipient, operative, and intensive care unit parameters to determine the relationship of filling pressure (central venous and pulmonary capillary wedge pressures) to prolonged mechanical ventilation and outcome. The mean central venous pressure was used to divide patients into high and low central venous pressure groups, which were then compared to determine differences in outcome and complication rates. RESULTS: A high central venous pressure was found to be associated with prolonged mechanical ventilation (odds ratio, 1.57; 95% confidence interval, 1.13-2.20; P = .008). After removing the effect of poor myocardial function by excluding patients with low cardiac index (< 2.2 L x min -1 x m(-2) ) and high inotrope requirement (> 10 microg/min), central venous pressure remained associated with prolonged mechanical ventilation (odds ratio, 2.31; 95% confidence interval, 1.31-4.07; P = .004). Duration of ventilation (P < .001), intensive care unit mortality (P = .02), hospital mortality (P = .09), and 2-month mortality (P = .02) were higher in patients with central venous pressures of greater than 7 mm Hg. There was no evidence of complications caused by hypovolemia in the low (< or = 7 mm Hg) central venous pressure group, who had lower inotrope requirements (P = .02) and lower creatinine levels (P = .013). Conclusions A high central venous pressure was associated with adverse outcomes after lung transplantation.

Successful mobilization of peripheral blood stem cells using recombinant human stem cell factor in heavily pretreated patients who have failed a previous attempt with a granulocyte colony-stimulating factor-based regimen.

To assess the efficacy of recombinant human stem cell factor (rHuSCF), 48 patients who had failed to mobilize >2.0 x 10(6) CD34+ cells/kg with granulocyte colony-stimulating factor (G-CSF) (10 microg/kg twice daily) with, or without, concomitant chemotherapy (G-CSF-based regimen), were remobilized with the addition of rHuSCF (20 microg/kg/day). In all, 18/48 (38%) achieved a total of >2.0 x 10(6) CD34+ cells/kg with the second rHuSCF-based mobilisation alone and 29/48 (60%) achieved a cumulative total of >2.0 x 10(6) CD34+ cells/kg following remobilization. Inclusion of chemotherapy in the mobilization regimen resulted in a higher yield of CD34+ cells/kg for both the initial G-CSF-based and subsequent rHuSCF-based regimens (0.90 vs 0.54, P < 0.01 and 2.36 vs 1.34, P < 0.01, respectively). The total peripheral blood stem cells PBSC collected from the G-CSF-based regimen, performance status, baseline platelet count and albumin were significantly associated with successful remobilization. Patients with multiple myeloma were also more likely to successfully remobilize. There was no threshold of total collected from the failed G-CSF-based regimen below which successful remobilization with the rHuSCF-based regimen was not possible. We therefore propose a predictive model [PBSC expected = 0.6+(G-CSF-based total collection)+2 (rHuSCF-based day 1 collection)] to calculate the cumulative total of PBSC expected following a maximum of five leukaphereses. This algorithm may permit the early identification of patients who are unlikely to achieve sufficient PBSC for transplantation and allow physicians to direct the resources involved in PBSC collection in a more appropriate and economical manner.

Chromosomal insertion of phenazine-1-carboxylic acid biosynthetic pathway enhances efficacy of damping-off disease control by Pseudomonas fluorescens.

A disarmed Tn5 vector (pUT::Ptac-phzABCDEFG) was used to introduce a single copy of the genes responsible for phenazine-1-carboxylic acid (PCA) biosynthesis into the chromosome of a plant-growth-promoting rhizobacterium Pseudomonas fluorescens. The PCA gene cluster was modified for expression under a constitutive Ptac promoter and lacked the phzIR regulators. PCA-producing variants significantly improved the ability of the wild-type P. fluorescens to reduce damping-off disease of pea seedlings caused by Pythium ultimum, even under conditions of heavy soil infestation. Under conditions of oxygen limitation that are typical of the rhizosphere, PCA production per cell in vitro was greater than that recorded in fast-growing, nutrient-rich cultures. Similarly, when the in vitro nutrient supply was limited, P fluorescens::phz variants that produced the most PCA effectively competed against P. ultimum by suppressing mycelial development. Soil-based bioassays confirmed that the level of PCA biosynthesis correlated directly with the efficacy of biological control and the persistence of inocula in soil microcosms. They also showed that soil pretreatment with bacteria provides a suitable method for plant protection by reducing infection, effectively decontaminating the soil. These data demonstrate that the insertion of a single chromosomal copy of the genes for a novel antifungal compound, PCA, enhances the ecological fitness of a natural isolate already adapted to the rhizosphere and capable of suppressing fungal disease.

Increased human cytomegalovirus (HCMV) DNA load in peripheral blood leukocytes after lung transplantation correlates with HCMV pneumonitis.

BACKGROUND: Human cytomegalovirus (HCMV) reactivation and disease remain relatively common in lung transplant recipients (LTR) despite the use of ganciclovir prophylaxis protocols for all HCMV at-risk patients. The specific aims of this study were to (1) describe the HCMV DNA viral load in the peripheral blood leukocytes (PBL) of a cohort of LTR during the first 6 months after lung transplantation; (2) prospectively determine whether HCMV DNA viral load predicts episodes of HCMV pneumonitis in LTR; and (3) study the effect of ganciclovir on HCMV viral load. METHODS: Competitive polymerase chain reaction using an internal standard and fluorometric detection were used to quantitate HCMV DNA in the PBL of a cohort of 26 LTR monthly for the first 6 months after transplantation (145 samples). All patients were treated with standard triple immunosuppression, and ganciclovir prophylaxis was given to all at-risk LTR (donor or recipient HCMV seropositive) for at least 8 weeks after transplantation. RESULTS: Thirteen episodes of histopathologically proven HCMV pneumonitis in nine subjects occurred during follow-up with a wide intra- and intersubject variation in the HCMV DNA PBL levels. HCMV detection had a sensitivity of 92% and specificity of 76% for HCMV pneumonitis (negative likelihood ratio, 9.5), whereas greater than 10-fold increases in HCMV DNA load had a specificity of 93% and sensitivity of 67% (positive likelihood ratio, 11). HCMV DNA detection had an adjusted odds ratio for HCMV pneumonitis of 107 (95% confidence interval, 14-821; P<0.005). In those with detectable HCMV DNA in PBL (n=44), HCMV DNA levels were 4.4 (95% confidence interval, 1.2-16.8) times higher in those with HCMV pneumonitis than in those without HCMV pneumonitis. Although ganciclovir treatment was very effective in treating HCMV pneumonitis and suppressing HCMV DNA levels, thrice weekly ganciclovir prophylaxis only partially controlled HCMV DNA levels and did not eliminate HCMV pneumonitis risk as three patients developed HCMV pneumonitis while on this regimen. CONCLUSIONS: HCMV DNA detection, absolute levels, and relative change from baseline in the PBL of LTR correlate with HCMV pneumonitis episodes and may be a useful intermediate outcome measure of the efficacy of ganciclovir prophylaxis and treatment strategies.