Melvyn Smith

Diagnosis of genital herpes by real time PCR in routine clinical practice

Background: Virus isolation in cell culture is the recognised diagnostic gold standard for genital herpes. Although increasing evidence indicates that polymerase chain reaction (PCR) provides a more rapid and sensitive diagnostic method, its implementation in routine diagnostic settings has been limited by concerns over contamination and cost. Objective: To evaluate the feasibility of replacing virus culture with PCR for the diagnosis of genital herpes in settings serving large populations of genitourinary medicine (GUM) attendees. Methods: Genital swabs collected from 233 consecutive GUM attendees with suspected genital herpes were tested in parallel by virus culture and automated real time PCR. Three specimen preparation methods were evaluated and the assay reliability was assessed by repeat testing, comparison with a commercially available assay, and herpes simplex virus (HSV) sequence analysis. Probe melting temperatures (Tm) were used to differentiate between HSV types without additional post-PCR steps. Results: HSV was detected in 79/233 (34%) samples by virus culture and 132/233 (57%) samples by PCR. PCR significantly increased HSV detection in both early (<5 days) and late (⩾5 days) presentations and in both first and recurrent episodes. HSV detection and typing by PCR was achieved within less than 4 hours leading to a significant reduction in labour compared to virus culture. Most specimens (120/132, 91%) were typed as HSV-2. Results were highly reproducible. Conclusions: Real time PCR is a highly reproducible, rapid, and labour efficient method for HSV detection in genital swabs. Its implementation is feasible in routine diagnostic settings.

Lung function prior to viral lower respiratory tract infections in prematurely born infants

Objective Prematurely born infants who develop respiratory syncytial virus (RSV) lower respiratory tract infections (LRTIs) have lung function abnormalities at follow-up. The aim of this study was to determine whether prematurely born infants who developed symptomatic RSV, or other viral LRTI(s), had poorer premorbid lung function than infants who did not develop LRTIs during the RSV season. Methods Lung function (functional residual capacity (FRC), compliance (Crs) and resistance (Rrs) of the respiratory system) was measured at 36 weeks postmenstrual age. After neonatal unit discharge, nasopharyngeal aspirates were obtained whenever the infants had an LRTI, regardless of whether this was in the community or in hospital. Nasopharyngeal aspirates were examined for RSV A and B, rhinovirus, influenza A and B, parainfluenza 1, 2 and 3, human metapneumovirus and adenovirus. Results 159 infants with a median gestational age of 34 (range 23–36) weeks were prospectively followed. 73 infants developed LRTIs: 27 had at least one RSV LRTI and 31 had at least one other viral LRTI, but not an RSV LRTI. Overall, there were no significant differences in the FRC (p=0.54), Crs (p=0.11) or Rrs (p=0.12) results between those who developed an RSV or other viral LRTI and those who did not develop an LRTI. Infants with RSV or other viral LRTIs who were admitted to hospital compared with those who were not had higher Rrs results (p=0.033 and p=0.039, respectively). Conclusion Diminished premorbid lung function may predispose prematurely born infants to severe viral LRTIs in infancy.

Lung function in prematurely born infants after viral lower respiratory tract infections

Background: Chronic respiratory morbidity has been reported in prematurely born infants after respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) lower respiratory tract infections (LRTIs). The aim of this study was to determine the impact of viral LRTI on lung function at follow-up of prematurely born infants. Methods: Fifty-nine infants born before 32 weeks of gestational age were prospectively followed after neonatal unit discharge. Nasopharyngeal aspirates were obtained when the infants developed LRTIs in hospital or the community. RSV was identified by immunofluorescence and/or culture. In addition, RSV and other viral infections were identified by real time reverse transcription polymerase chain reaction. At a corrected age of 1 year, measurements of lung volume [functional residual capacity (FRC)pleth] and airway resistance (Raw) were made by plethysmography, and lung volume was also assessed by helium gas dilution (FRCHe). Before the measurements, parents completed diary cards for 1 month documenting on a daily basis whether their infant wheezed, coughed, or required bronchodilator therapy. Results: Twenty-five infants had at least 1 proven RSV LRTI (RSV-positive group). The RSV-positive group compared with the rest of the cohort had similar lung volumes, but significantly higher Raw (P = 0.002), more days of wheeze (P < 0.001), and bronchodilator requirement (P = 0.027). Regression analysis also identified that hMPV LRTI was associated with elevated airways resistance at follow-up. Conclusion: RSV and hMPV LRTIs in prematurely born infants are associated with abnormal lung function at follow-up.

A comprehensive diagnostic approach using galactomannan, targeted β-d-glucan, baseline computerized tomography and biopsy yields a significant burden of invasive fungal disease in at risk haematology patients

Invasive fungal disease (IFD) is difficult to diagnose. We investigated the incidence of IFD and risk factors using the revised European Organization for Research and Treatment of Cancer (EORTC) and the Mycoses Study Group (MSG) definitions. Patients (N = 203) undergoing intensive therapy with expected neutropenia ≥10 d were recruited prospectively and followed for a median (range) of 556 (12–730) d. Baseline chest computerized tomography (CT) was performed pre‐therapy. Twice‐weekly surveillance with galactomannan (GM) was combined with targeted β‐d‐glucan (BDG) testing on patients with possible IFD or who were GM‐positive. Tissue diagnosis was obtained whenever possible. The cumulative incidence of proven/probable IFD among the 202 evaluable cases after 2 years follow‐up was 21%, including 14 proven and 30 probable IFDs. Using either GM or BDG as the sole biomarker (plus host and clinical evidence) the apparent overall incidence of proven/probable IFD was 11% and 16%, respectively. Combined GM/BDG detected all biopsy‐proven mould IFD. Baseline CT abnormalities were found in 76/202 (38%) patients. Baseline CT abnormalities and Karnofsky score <90, monocytopenia >10 d and bacteraemia were independent risk factors associated with greater than twofold increased IFD risk. This combined diagnostic approach identified a high incidence of IFD and important risk factors in this cohort.

Herpes Simplex Virus Type 2 (HSV-2) Seroprevalence at the Time of HIV-1 Diagnosis and Seroincidence After HIV-1 Diagnosis in an Ethnically Diverse Cohort of HIV-1-Infected Persons

Objective: The objective of this study was to determine herpes simplex virus type 2 (HSV-2) seroprevalence at HIV-1 diagnosis and seroincidence ≥1 year after HIV-1 diagnosis. Methods: HSV type-specific antibodies were detected by enzyme immunoassay. Results: The cohort comprised 850 adults diagnosed HIV-positive in 1986–2001 and followed for a median of 3 years. HSV-2 seroprevalence was 63% (95% confidence interval [CI], 60–66%) and was associated with female gender, heterosexual risk group, black ethnicity, and older age. HSV-2 seroincidence was 1.8 per 100 person-years (95% CI, 0.8–2.8) and was associated with other sexually transmitted diseases, including human papilloma virus infection (P = 0.005) and gonorrhea (P = 0.05). A diagnosis of genital herpes was made in 21% HSV-2-seropositive persons and was more likely in those who tested HIV-positive before 1997 (adjusted odds ratio, 5.11; 95% CI, 3.28–7.98; P = 0.0001). Conclusions: Results confirm the epidemiologic association between HIV-1 and HSV-2. HSV-2 seroconversion was a marker of high-risk sexual behavior. The likelihood of developing symptoms of genital herpes declined from 1997 onward.

Respiratory outcome of prematurely born infants following human rhinovirus A and C infections.

Human rhinoviruses (HRVs) are a common cause of lower respiratory tract infections (LRTIs) and are associated with chronic respiratory morbidity. Our aim was to determine whether HRV species A or C were associated with chronic respiratory morbidity and increased health care utilisation in prematurely born infants. A number of 153 infants with a median gestational age of 34 (range 23–35) weeks were prospectively followed. Nasopharyngeal aspirates were collected whenever the infants had LRTIs regardless of hospitalisation status. Parents completed a respiratory diary card and health questionnaire about their infant when they were 11 and 12 months corrected age, respectively. The health-related cost of care during infancy was calculated from the medical records using the National Health Service (NHS) reference costing scheme and the British National Formulary for children. There were 32 infants that developed 40 HRV LRTIs; samples were available from 23 of the 32 infants for subtyping. Nine infants had HRV-A LRTIs, 13 HRV-C LRTIs, and one infant had a HRV-B LRTI. Exclusion of infants who also had RSV LRTIs revealed that the infants who had a HRV-C LRTI were more likely to wheeze (p < 0.0005) and use respiratory medications (p < 0.0005) and had more days of wheeze (p = 0.01) and used an inhaler (p = 0.02) than the no LRTI group. In addition, the respiratory cost of care was greater for the HRV-C LRTI than the no LRTI group (p < 0.0005). Conclusion: Our results suggest HRV-C is associated with chronic respiratory morbidity during infancy in prematurely born infants.

Rhinovirus infection and healthcare utilisation in prematurely born infants

Our aim was to determine whether rhinovirus (RV) lower respiratory tract infections (LRTIs) in prematurely born infants increase health-related cost of care during infancy. 153 infants born at <36 weeks of gestation were prospectively followed to 1 year. Cost of care was calculated from the National Health Service reference costing scheme and healthcare utilisation determined by examining hospital/general practitioner records. 20 infants developed RV LRTIs (RV group), 17 respiratory syncytial virus (RSV) LRTIs (RSV group), 12 both RV and RSV LRTIs (RV/RSV group) and 74 had no LRTI (no LRTI group). Compared with the no LRTI group, the RV/RSV LRTI group had the greatest increase in adjusted mean cost (difference GBP 5769), followed by the RV LRTI group (difference GBP 278) and, finally, the RSV LRTI group (difference GBP 172) (p=0.045). The RV group had more outpatient (p<0.05) and respiratory-related general practitioner (p<0.05) attendances, more wheezed at follow-up (p<0.001) than the no LRTI group and more had respiratory-related outpatient attendances than the RSV LRTI group (p<0.05). We conclude that RV LRTIs were associated with increased health-related cost of care during infancy; our results suggest that the RV group compared with the RSV group suffered greater chronic respiratory morbidity. Rhinovirus LRTIs in prematurely born infants increases health-related cost of care during infancy http://ow.ly/mxyZH

Granulocyte-macrophage colony-stimulating factor enhances viral load in human brain tissue: Amelioration with stavudine

Background Granulocyte-macrophage colony-stimulating factor (GM-CSF) is elevated in cerebrospinal fluid in HIV- associated dementia; in addition, therapeutic GM-CSF elevates plasma viral load. Objective To assess the effect of GM-CSF on viral replication and the potential ameliorative effect of antiretroviral therapy. Design A primary human brain aggregate system is used as a model of the in vivo situation. Method Cultured aggregates were infected with the macrophage tropic strain HIV-1SF162 and then exposed to varying GM-CSF concentrations and 0.3 μmol/l stavudine. Viral replication was assessed by p24 expression in the supernatant and aggregates. Immunohistochemistry identified neurons, astrocytes, microglia and oligodendrocytes. Results A GM-CSF concentration of 1 ng/ml resulted in a fivefold increase in microglial cells, the main HIV cellular reservoir (P = 0.0001). Prior GM-CSF exposure before infection of the aggregates resulted in sixfold increase in p24 levels compared with non-GM-CSF-exposed infected aggregates. Infected aggregates with or without GM-CSF had significant neuronal loss of 50% and 45%, respectively, and astrocytosis. Addition of stavudine to the infected aggregates, even in the presence of GM-CSF, reduced p24 levels to zero and prevented neuronal loss and astrocytosis. Conclusions This study demonstrates that GM-CSF enhances viral replication while addition of stavudine prevents this potentially detrimental process.

Disseminated neonatal herpes simplex virus (HSV) type 2 infection diagnosed by HSV DNA detection in blood and successfully managed by liver transplantation

We report a case of neonatal herpes presenting with liver failure and disseminated coagulopathy which followed unrecognised maternal primary genital herpes and was diagnosed by herpes simplex virus DNA detection in blood by polymerase chain reaction 2 weeks after initiation of empiric intravenous aciclovir. The child underwent liver transplantation while receiving suppressive antiviral therapy and remains well after 10 months of follow-up. Conclusion:our case highlights potential pitfalls in the diagnosis of neonatal herpes and indicates a role for blood herpes simplex virus polymerase chain reaction as a sensitive diagnostic tool in disseminated infection. It is one of very few reports where liver transplantation has been successfully carried out in a neonate with herpes simplex virus-induced liver failure.

Adjunctive mecA PCR for Routine Detection of Methicillin Susceptibility in Clinical Isolates of Coagulase-Negative Staphylococci

ABSTRACT Concerns over the reliability of routine sensitivity testing in coagulase-negative staphylococci often lead to the use of potentially less-effective antibiotics as few laboratories have access to routine tests for the mecA resistance gene. Although previous studies have shown a reasonable correlation between oxacillin disc and automated sensitivity testing, changing epidemiology and methodology dictate periodic reappraisal of these methods. In the present study, we evaluated two real-time PCR assays against novel targets in the mecA gene as an adjunct to routine susceptibility testing using the Vitek II AST-P620 card. All samples were further examined for the presence of the mecC gene. Of 118 strains of coagulase-negative staphylococci tested, 81 were oxacillin resistant and 37 oxacillin susceptible by the Vitek II assay compared with 103 positive and 15 negative by mecA PCR. In-house PCR results correlated well with a previously published reference PCR, though little correlation was found between mecA PCR or Vitek II and PBP 2a latex agglutination. Incubation conditions may have affected the accuracy of the latter test. None of the strains tested were mecC PCR positive. The inclusion of dual-target PCRs in the testing algorithm was inexpensive and offered the safest strategy for determining beta-lactam susceptibility in coagulase-negative staphylococci in our laboratory.