Tony Mazzulli

Clinical utility of quantitative cytomegalovirus viral load determination for predicting cytomegalovirus disease in liver transplant recipients

BACKGROUND The early detection of cytomegalovirus (CMV) after liver transplantation may form the basis of a preemptive strategy for prevention of active CMV disease. METHODS We prospectively analyzed the clinical use of weekly quantitative polymerase chain reaction-(PCR) based plasma viral load determinations and the antigenemia assay for predicting the development of active CMV disease in 97 consecutive liver transplant recipients. RESULTS CMV disease occurred in 21/97 patients. Using a positive cut-off of >400 copies/ml plasma, PCR had a sensitivity of 100%, specificity 47.4%, positive predictive value 34.4% and negative predictive value 100% for prediction of CMV disease. Respective values for a positive antigenemia (>0 positive cells/slide) were 95.2, 55.3, 37.0, and 97.7%. Different cut-off points for a positive test were analyzed using receiver-operating characteristic (ROC) curves. The optimal cut-off for viral load was in the range of 2000-5000 copies/ml (sensitivity 85.7%, specificity 86.8%, PPV 64.3%, NPV 95.7% for >5000 copies/ml). The optimal cut-off for antigenemia was in the range of four to six positive cells/slide. Mean peak viral load in symptomatic patients was 73,715 copies per/ml versus 3615 copies/ml in patients with asymptomatic CMV reactivation (P<0.001). In a multivariate logistic regression analysis of risk factors for CMV disease (CMV serostatus, acute rejection, and induction immunosuppression), peak viral load and peak antigenemia emerged as the only significant independent predictors of CMV disease (for PCR, odds ratio=1.40/1000 copy/ml increase in viral load, P=0.0001; for antigenemia odds ratio=1.17/1 positive cell/slide). CONCLUSIONS Plasma viral load by quantitative PCR is useful for predicting CMV disease and could be used in a preemptive strategy.

Negative mucosal synergy between Herpes simplex type 2 and HIV in the female genital tract

Objective:There is substantial epidemiological evidence that infection by Herpes simplex virus type 2 (HSV2) enhances both HIV susceptibility and subsequent sexual transmission. Both infections are extremely common in female sex workers (FSWs) in sub-Saharan Africa, and up to 80% of new HIV infections in urban men in the region are acquired via transactional sex. The present study aimed to elucidate the mucosal immune interactions between HIV and HSV2 in the genital tract. Methods:Endocervical immune cell populations, cytokine/chemokine protein levels in cervico-vaginal secretions and cervical immune gene expression profiles were measured in a well-defined cohort of HIV-infected and uninfected Kenyan FSWs. Associations between the genital immune milieu and infection by and/or shedding of common genital co-pathogens were examined. Results:HIV-infected FSWs were much more likely to be infected by HSV2, and to shed HSV2 DNA in the genital tract. There was also a profound negative ‘mucosal synergy’ between these viruses. In HIV uninfected FSWs, HSV2 infection was associated with a ten-fold increase in cervical immature dendritic cells (iDC) expressing DC-SIGN, and a three-fold increase in cervical CD4+ T cells expressing CCR5. HIV infection was associated with iDC depletion in the cervix, and with increased HSV2 genital reactivation, which in turn was associated with HIV shedding levels. Conclusions:The findings suggest a mucosal vicious circle in which HSV2 infection increases HIV target cells in the genital mucosa, subsequent HIV infection impairs HSV2 mucosal immune control, and local HSV2 reactivation enhances both HSV2 and HIV transmission.

A Canadian National Surveillance Study of Urinary Tract Isolates from Outpatients: Comparison of the Activities of Trimethoprim-Sulfamethoxazole, Ampicillin, Mecillinam, Nitrofurantoin, and Ciprofloxacin

ABSTRACT Ampicillin, trimethoprim-sulfamethoxazole, mecillinam, nitrofurantoin, and ciprofloxacin mean resistance rates for 2,000 urinary tract isolates collected from outpatients across Canada in 1998 were 41.1, 19.2, 14.7, 5.0, and 1.8%, respectively. ForEscherichia coli isolates alone (n = 1,681) comparable rates were 41.0, 18.9, 7.4, 0.1, and 1.2%, respectively. The majority of E. coli isolates resistant to ampicillin, trimethoprim-sulfamethoxazole, or ciprofloxacin were susceptible (MIC, <16 μg/ml) to mecillinam.

A metagenomic analysis of pandemic influenza A (2009 H1N1) infection in patients from North America.

Although metagenomics has been previously employed for pathogen discovery, its cost and complexity have prevented its use as a practical front-line diagnostic for unknown infectious diseases. Here we demonstrate the utility of two metagenomics-based strategies, a pan-viral microarray (Virochip) and deep sequencing, for the identification and characterization of 2009 pandemic H1N1 influenza A virus. Using nasopharyngeal swabs collected during the earliest stages of the pandemic in Mexico, Canada, and the United States (n = 17), the Virochip was able to detect a novel virus most closely related to swine influenza viruses without a priori information. Deep sequencing yielded reads corresponding to 2009 H1N1 influenza in each sample (percentage of aligned sequences corresponding to 2009 H1N1 ranging from 0.0011% to 10.9%), with up to 97% coverage of the influenza genome in one sample. Detection of 2009 H1N1 by deep sequencing was possible even at titers near the limits of detection for specific RT-PCR, and the percentage of sequence reads was linearly correlated with virus titer. Deep sequencing also provided insights into the upper respiratory microbiota and host gene expression in response to 2009 H1N1 infection. An unbiased analysis combining sequence data from all 17 outbreak samples revealed that 90% of the 2009 H1N1 genome could be assembled de novo without the use of any reference sequence, including assembly of several near full-length genomic segments. These results indicate that a streamlined metagenomics detection strategy can potentially replace the multiple conventional diagnostic tests required to investigate an outbreak of a novel pathogen, and provide a blueprint for comprehensive diagnosis of unexplained acute illnesses or outbreaks in clinical and public health settings.

Clinical Utility of Cytomegalovirus (CMV) Serology Testing in High‐risk CMV D+/R− Transplant Recipients

Late‐onset cytomegalovirus (CMV) disease is a significant problem in D+/R− solid organ transplant (SOT) patients who receive antiviral prophylaxis. We assessed the clinical utility of CMV IgG and IgM serology testing for predicting late‐onset CMV disease. We evaluated 352 D+/R− transplant recipients who participated in a trial comparing 100 days of ganciclovir versus valganciclovir prophylaxis. CMV serology was assessed on day 28, 56, 100, and 6 and 12 months post‐transplant. IgG seroconversion occurred in 26.9% of patients by day 100, and in 63.4% and 75.3% by 6 and 12 months, respectively. IgM seroconversion occurred in 8.3%, 41.8% and 54.9% by day 100, month 6 and month 12, respectively. Seroconversion by day 100 (end of prophylaxis) was not predictive of subsequent CMV disease (CMV disease 13.3% if seropositive vs. 17.8% if seronegative; p = NS). However, at 6 months post‐transplant, IgG serostatus was predictive of subsequent CMV disease between month 6 and 12 (CMV disease 1.3% if seropositive vs. 10.0% if seronegative; p = 0.002). In D+/R− patients, CMV serology testing is for the most part not clinically useful for predicting subsequent disease. However, seroconversion by 6 months may be useful for identifying patients at risk of late‐onset CMV disease.

Clinical impact of human herpesvirus 6 infection after liver transplantation.

BACKGROUND Reactivation of human herpesvirus 6 (HHV-6) appears to be common after transplant. Viral reactivation may result in febrile illness and may also play an immunomodulatory role that leads to indirect effects such as opportunistic infections and rejection. The objective of this study was to determine the clinical impact of HHV-6 infection after liver transplantation including both direct and indirect effects. METHODS This was a prospective single center cohort study of 200 consecutive patients undergoing liver transplantation. Systemic serial HHV-6 viral load measurements and all clinical outcomes including development of opportunistic infections, cytomegalovirus (CMV) disease, and rejection were determined. RESULTS HHV-6 infection (defined as viral load > or = 2 log10 copies/microg input DNA) occurred in 56/200 (28%) patients. Symptomatic disease attributable to HHV-6 alone occurred in 2/200 (1%) patients. Univariate analysis revealed HHV-6 infection was associated with the development of opportunistic infection and CMV disease. In a multivariate analysis designed to control for the level of immunosuppression, the risk of opportunistic infection increased by 3.68-fold in patients with HHV-6 infection (95% confidence interval [CI], 1.86-7.27; P=0.001). In a similar multivariate analysis, the risk of CMV disease increased by 3.59-fold in patients with HHV-6 infection (95% CI, 1.53-8.44; P=0.003). HHV-6 infection was not associated with rejection except in the subgroup of patients with rejection after 30 days posttransplant (odds ration 2.27; 95% CI, 1.09-4.77; P=0.029). CONCLUSIONS HHV-6 reactivation after transplant is common and is associated with the development of opportunistic infections and CMV disease and possibly with a subgroup of acute rejection episodes. HHV-6 infection likely has a significant impact in transplant recipients through indirect effects of viral replication.

Human Herpesvirus—6 Is Associated with Cytomegalovirus Reactivation in Liver Transplant Recipients

Human herpesvirus-6 (HHV-6) may be a risk factor for cytomegalovirus (CMV) disease in posttransplant patients, possibly through a direct interaction or through a general immunomodulatory effect. To examine this possibility, 88 liver transplant recipients were monitored with serial HHV-6 polymerase chain reaction (PCR), CMV antigenemia, and CMV plasma viral load. HHV-6 infection was defined by a positive PCR of peripheral blood lymphocytes. Forty-eight (54.4%) of 88 patients had at least 1 positive HHV-6 PCR. CMV recurrence was significantly more common in patients with HHV-6 infection (38/48 patients [79. 2%]), compared with recurrence in those without HHV-6 infection (18/40 patients [45%]; P=.001). Peak CMV viral load was 24, 147+/-6799 copies/mL in patients with HHV-6 infection versus 8391+/-4598 copies/mL in patients without HHV-6 infection (P=.001). Symptomatic CMV disease was more common in patients with HHV-6 infection than it was in those without infection (15/48 patients [31. 3%] vs. 4/10 patients [10.0%]; P=.013). In a multivariate analysis including other risk factors for CMV, HHV-6 infection remained an independent risk factor for CMV disease (P=.013; odds ratio, 7.26; 95% confidence interval, 1.52-34.72). HHV-6 is associated with CMV infection and disease, thus supporting an interaction between these viruses.

Antimicrobial resistance among clinical isolates of Streptococcus pneumoniae in Canada during 2000.

ABSTRACT A total of 2,245 clinical isolates of Streptococcus pneumoniae were collected from 63 microbiology laboratories from across Canada during 2000 and characterized at a central laboratory. Of these isolates, 12.4% were not susceptible to penicillin (penicillin MIC, ≥0.12 μg/ml) and 5.8% were resistant (MIC, ≥2 μg/ml). Resistance rates among non-β-lactam agents were the following: macrolides, 11.1%; clindamycin, 5.7%; chloramphenicol, 2.2%; levofloxacin, 0.9%; gatifloxacin, 0.8%; moxifloxacin, 0.4%; and trimethoprim-sulfamethoxazole, 11.3%. The MICs at which 90% of the isolates were inhibited (MIC90s) of the fluoroquinolones were the following: gemifloxacin, 0.03 μg/ml; BMS-284756, 0.06 μg/ml; moxifloxacin, 0.12 μg/ml; gatifloxacin, 0.25 μg/ml; levofloxacin, 1 μg/ml; and ciprofloxacin, 1 μg/ml. Of 578 isolates from the lower respiratory tract, 21 (3.6%) were inhibited at ciprofloxacin MICs of ≥4 μg/ml. None of the 768 isolates from children were inhibited at ciprofloxacin MICs of ≥4 μg/ml, compared to 3 of 731 (0.6%) from those ages 15 to 64 (all of these >60 years old), and 27 of 707 (3.8%) from those over 65. The MIC90s for ABT-773 and telithromycin were 0.015 μg/ml for macrolide-susceptible isolates and 0.12 and 0.5 μg/ml, respectively, for macrolide-resistant isolates. The MIC of linezolid was ≤2 μg/ml for all isolates. Many of the new antimicrobial agents tested in this study appear to have potential for the treatment of multidrug-resistant strains of pneumococci.

Interactions Between Cytomegalovirus, Human Herpesvirus‐6, and the Recurrence of Hepatitis C After Liver Transplantation

Recurrence of hepatitis C (HCV) following liver transplantation is common. Herpesvirus reactivation following transplant may have an immunomodulatory effect resulting in increased HCV replication. We studied whether cytomegalovirus (CMV) and human herpesvirus‐6 (HHV‐6) may be associated with HCV recurrence and viral load after transplant. We prospectively followed 66 HCV liver‐transplant recipients with serial viral load testing for CMV and HHV‐6. Infection and viral load were correlated with the development of biopsy‐proven HCV recurrence and HCV viral loads. Histologic recurrence of HCV occurred in 41/66 (62.1%) patients. In the primary analysis, CMV infection and disease, and HHV‐6 infection were not associated with HCV recurrence. Peak CMV and HHV‐6 viral loads were not significantly different in patients with and without recurrence. No correlation was observed between HCV viral loads at 1 and 3 months post‐transplant and peak HHV‐6 or CMV viral loads. In a subgroup analysis, HHV‐6 infection was associated with the development of more severe recurrence (hepatitis and/or fibrosis score ≥ 2) (p = 0.01). Also, fibrosis scores at last follow up were higher in patients with CMV disease (1.67 vs. 0.56; p = 0.016) and in patients with HHV‐6 infection (1.18 vs. 0.55; p = 0.031). In conclusion, HHV‐6 and CMV infection and viral load were not associated with increased overall rates of HCV recurrence or HCV viral load after liver transplantation but may be associated with more severe forms of recurrence.

Effects of 630-, 660-, 810-, and 905-nm laser irradiation delivering radiant exposure of 1-50 J/cm2 on three species of bacteria in vitro.

OBJECTIVE To examine the effects of low-intensity laser therapy (LILT) on bacterial growth in vitro. BACKGROUND DATA LILT is undergoing investigation as a treatment for accelerating healing of open wounds. The potential of coincident effects on wound bacteria has received little attention. Increased bacterial proliferation could further delay recovery; conversely inhibition could be beneficial. MATERIALS AND METHODS Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus were plated on agar and then irradiated with wavelengths of 630, 660, 810, and 905 nm (0.015 W/cm(2)) and radiant exposures of 1-50 J/cm(2). In addition, E. coli was irradiated with 810 nm at an irradiance of 0.03 W/cm(2) (1-50 J/cm(2)). Cells were counted after 20 h of incubation post LILT. Repeated measures ANOVA and Tukey adjusted post hoc tests were used for analysis. RESULTS There were interactions between wavelength and species (p = 0.0001) and between wavelength and radiant exposure (p = 0.007) in the overall effects on bacterial growth; therefore, individual wavelengths were analyzed. Over all types of bacteria, there were overall growth effects using 810- and 630-nm lasers, with species differences at 630 nm. Effects occurred at low radiant exposures (1-20 J/cm(2)). Overall effects were marginal using 660 nm and negative at 905 nm. Inhibition of P. aeruginosa followed irradiation using 810 nm at 5 J/cm(2) (-23%; p = 0.02). Irradiation using 630 nm at 1 J/cm(2) inhibited P. aeruginosa and E. coli (-27%). Irradiation using 810 nm (0.015 W/cm(2)) increased E. coli growth, but with increased irradiance (0.03 W/cm(2)) the growth was significant (p = 0.04), reaching 30% at 20 J/cm(2) (p = 0.01). S. aureus growth increased 27% following 905-nm irradiation at 50 J/cm(2). CONCLUSION LILT applied to wounds, delivering commonly used wavelengths and radiant exposures in the range of 1-20 J/cm(2), could produce changes in bacterial growth of considerable importance for wound healing. A wavelength of 630 nm appeared to be most commonly associated with bacterial inhibition. The findings of this study might be useful as a basis for selecting LILT for infected wounds.