Zin Naing

Multiplex PCR Testing Detection of Higher-than-Expected Rates of Cervical Mycoplasma, Ureaplasma, and Trichomonas and Viral Agent Infections in Sexually Active Australian Women

ABSTRACT Knowing the prevalence of potential etiologic agents of nongonococcal and nonchlamydial cervicitis is important for improving the efficacy of empirical treatments for this commonly encountered condition. We describe four multiplex PCRs (mPCRs), designated VDL05, VDL06, VDL07, and VDL09, which facilitate the detection of a wide range of agents either known to be or putatively associated with cervicitis, including cytomegalovirus (CMV), enterovirus (EV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), herpes simplex virus type 1 (HSV-1), and herpes simplex virus type 2 (HSV-2) (VDL05); Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma genitalium, and Mycoplasma hominis (VDL06); Chlamydia trachomatis, Trichomonas vaginalis, Treponema pallidum, and group B streptococci (VDL07); and adenovirus species A to E (VDL09). The mPCRs were used to test 233 cervical swabs from 175 women attending a sexual-health clinic in Sydney, Australia, during 2006 and 2007. The agents detected alone or in combination in all cervical swabs (percentage of total swabs) included CMV (6.0), EV (2.1), EBV (2.6), VZV (4.7), HSV-1 (2.6), HSV-2 (0.8), HSV-2 and VZV (0.4), U. parvum (57.0), U. urealyticum (6.1), M. genitalium (1.3), M. hominis (13.7), C. trachomatis (0.4), T. vaginalis (3.4), and group B streptococci (0.4). Adenovirus species A to E and T. pallidum were not detected. These assays are adaptable for routine diagnostic laboratories and provide an opportunity to measure the true prevalence of microorganisms potentially associated with cervicitis and other genital infections.

Prevention of congenital cytomegalovirus complications by maternal and neonatal treatments: a systematic review.

Human cytomegalovirus is the leading non‐genetic cause of congenital malformation in developed countries. Congenital CMV may result in fetal and neonatal death or development of serious clinical sequelae. In this review, we identified evidence‐based interventions for prevention of congenital CMV at the primary level (prevention of maternal infection), secondary level (risk reduction of fetal infection and disease) and tertiary level (risk reduction of infected neonates being affected by CMV). A systematic review of existing literature revealed 24 eligible studies that met the inclusion criteria. Prevention of maternal infection using hygiene and behavioural interventions reduced maternal seroconversion rates during pregnancy. However, evidence suggested maternal adherence to education on preventative behaviours was a limiting factor. Treatment of maternal CMV infection with hyperimmune globulin (HIG) showed some evidence for efficacy in prevention of fetal infection and fetal/neonatal morbidity with a reasonable safety profile. However, more robust clinical evidence is required before HIG therapy can be routinely recommended. Limited evidence also existed for the safety and efficacy of established CMV antivirals (valaciclovir, ganciclovir and valganciclovir) to treat neonatal consequences of CMV infection, but toxicity and lack of randomised clinical trial data remain major issues. In the absence of a licensed CMV vaccine or robust clinical evidence for anti‐CMV therapeutics, patient education and behavioural interventions that emphasise adherence remain the best preventative strategies for congenital CMV. There is a strong need for further data on the use of HIG and other antivirals in pregnancy, as well as the development of less toxic, novel, antiviral agents. Copyright

Serological and virological investigation of the role of the herpesviruses EBV, CMV and HHV-6 in post-infective fatigue syndrome.

Multiple previous studies have sought evidence for ongoing, active infection with, or reactivation of, Herpesviruses in patients with chronic fatigue syndrome (CFS), with conflicting results. This study aimed to clarify this by studying 20 patients enrolled in a well‐characterized model of the onset and evolution of CFS, the prospective cohort of the Dubbo Infection Outcomes Study (DIOS). The patients selected for examination included five CFS patients with primary Epstein–Barr virus (EBV) infection; five CFS patients with acute viral infection not caused by EBV; and 10 matched controls with prompt resolution of primary EBV infection. Serum samples from three timepoints were assayed using a comprehensive range of serological assays for EBV, HHV‐6, and CMV. Viral genomes were assessed using quantitative PCR assays. All patients were seropositive for HHV‐6, and 10 were seropositive for CMV at infection baseline (five patients and five controls). Low titer CMV IgM antibodies were found at infection baseline in two of these cases and three control patients. HHV‐6 IgG antibody titers were highest at infection baseline but did not differ between the CFS cases and the control patients. There were increases in EBV IgG VCA p18, EBNA‐1 IgG, and EA IgG titers over time, but these did not differ between CFS cases and control patients. EBV and HHV6 DNA levels were at control levels in a minority of samples, and CMV was undetectable in all samples. These data do not support the hypothesis of ongoing or reactivated EBV, HHV‐6, or CMV infection in the pathogenesis of CFS. J. Med. Virol. 82:1684–1688, 2010. 2010 Wiley‐Liss, Inc.

Congenital cytomegalovirus infection in pregnancy: a review of prevalence, clinical features, diagnosis and prevention

Human cytomegalovirus (CMV) is under‐recognised, despite being the leading infectious cause of congenital malformation, affecting ~0.3% of Australian live births. Approximately 11% of infants born with congenital CMV infection are symptomatic, resulting in clinical manifestations, including jaundice, hepatosplenomegaly, petechiae, microcephaly, intrauterine growth restriction and death. Congenital CMV infection may cause severe long‐term sequelae, including progressive sensorineural hearing loss and developmental delay in 40–58% of symptomatic neonates, and ~14% of initially asymptomatic infected neonates. Up to 50% of maternal CMV infections have nonspecific clinical manifestations, and most remain undetected unless specific serological testing is undertaken. The combination of serology tests for CMV‐specific IgM, IgG and IgG avidity provide improved distinction between primary and secondary maternal infections. In pregnancies with confirmed primary maternal CMV infection, amniocentesis with CMV‐PCR performed on amniotic fluid, undertaken after 21–22 weeks gestation, may determine whether maternofetal virus transmission has occurred. Ultrasound and, to a lesser extent, magnetic resonance imaging are valuable tools to assess fetal structural and growth abnormalities, although the absence of fetal abnormalities does not exclude fetal damage. Diagnosis of congenital CMV infection at birth or in the first 3 weeks of an infants life is crucial, as this should prompt interventions for prevention of delayed‐onset hearing loss and neurodevelopmental delay in affected infants. Prevention strategies should also target mothers because increased awareness and hygiene measures may reduce maternal infection. Recognition of the importance of CMV in pregnancy and in neonates is increasingly needed, particularly as therapeutic and preventive interventions expand for this serious problem.

Human cytomegalovirus-induces cytokine changes in the placenta with implications for adverse pregnancy outcomes.

Human cytomegalovirus (CMV) infection of the developing fetus can result in adverse pregnancy outcomes including death in utero. Fetal injury results from direct viral cytopathic damage to the CMV-infected fetus, although evidence suggests CMV placental infection may indirectly cause injury to the fetus, possibly via immune dysregulation with placental dysfunction. This study investigated the effects of CMV infection on expression of the chemokine MCP-1 (CCL2) and cytokine TNF-α in placentae from naturally infected stillborn babies, and compared these changes with those found in placental villous explant histocultures acutely infected with CMV ex vivo. Tissue cytokine protein levels were assessed using quantitative immunohistochemistry. CMV-infected placentae from stillborn babies had significantly elevated MCP-1 and TNF-α levels compared with uninfected placentae (p = 0.001 and p = 0.007), which was not observed in placentae infected with other microorganisms (p = 0.62 and p = 0.71) (n = 7 per group). Modelling acute clinical infection using ex vivo placental explant histocultures showed infection with CMV laboratory strain AD169 (0.2 pfu/ml) caused significantly elevated expression of MCP-1 and TNF-α compared with uninfected explants (p = 0.0003 and p<0.0001) (n = 25 per group). Explant infection with wild-type Merlin at a tenfold lower multiplicity of infection (0.02 pfu/ml), caused a significant positive correlation between increased explant infection and upregulation of MCP-1 and TNF-α expression (p = 0.0001 and p = 0.017). Cytokine dysregulation has been associated with adverse outcomes of pregnancy, and can negatively affect placental development and function. These novel findings demonstrate CMV infection modulates the placental immune environment in vivo and in a multicellular ex vivo model, suggesting CMV-induced cytokine modulation as a potential initiator and/or exacerbator of placental and fetal injury.

Enterovirus infection induces cytokine and chemokine expression in insulin-producing cells.

Despite evidence supporting an association between enterovirus (EV) infection and type 1 diabetes, the etiological mechanism(s) for EV‐induced beta cell destruction is(are) not well understood. In this study, the effects of Coxsackievirus B (CVB) 1–6 on cell lysis and cytokine/chemokine expression in the insulinoma‐1 (INS‐1) beta cell line were investigated. Cytolysis was assessed using tissue culture infectious dose 50 (TCID50). Quantitative RT‐PCR was used to measure viral RNA and mRNA of cytokines interferon (IFN)‐α, IFN‐β, IFN‐γ, tumor necrosis factor (TNF)‐α, and chemokine (C–X–C motif) ligand 10 (CXCL10), chemokine (C–C motif) ligand 2 (CCL2), and chemokine (C–C motif) ligand 5 (CCL5) in infected INS‐1 cells. CVB2, 4, 5, and 6 lysed and replicated in INS‐1 cells; TCID50 was lowest for CVB5 and highest for CVB6. IFN‐γ, CXCL10, and CCL5 mRNA levels all increased significantly following infection with CVB2, 4, 5, and 6 (P < 0.05). CCL2 mRNA increased with CVB2, 5, and 6 (P < 0.05), IFN‐α mRNA increased with CVB5 infection (P < 0.05), while TNF‐α mRNA and IFN‐β mRNA (P < 0.001) increased with CVB2 infection. Dose‐dependent effects of infection on cytokine mRNA levels were observed for all (P < 0.01) except IFN‐γ. Following inoculation of INS‐1 cells with CVB1 and 3, viral RNA was not detected and cytokine/chemokine mRNA levels were unchanged. In conclusion, CVB2, 4, 5, and 6 induce dose‐dependent cytokine and chemokine mRNA production from INS‐1 cells suggesting that pro‐inflammatory cytokine and chemokine secretion by beta cells is a potential mechanism for EV‐induced beta cell damage in type 1 diabetes. J. Med. Virol. 82:1950–1957, 2010.

Trichomonas vaginalis: underdiagnosis in urban Australia could facilitate re-emergence

Objectives Trichomonas vaginalis (TV) has a low profile in urban sexually transmitted infection (STI) clinics in many developed countries. The objective of this study was to determine the true prevalence of TV in an Australian urban sexual health setting using sensitive molecular diagnostic techniques. Methods A cross-sectional study investigating the aetiology of cervicitis in women attending two urban sexual health clinics in Sydney, Australia, enrolled 356 consecutive eligible women from 2006 to 2008. The diagnostic yield from the standard clinical practice of discretionary high vaginal wet preparation microscopy in women with suspicious vaginal discharge was compared with universal use of nested PCR for TV of cervical samples. Results TV was detected by PCR in 17/356 women (4.8%, 95% CI 2.8 to 7.5%), whereas only four cases (1.1%, 95% CI 0.3 to 2.8%) were detected by discretionary wet preparation microscopy. Eleven of the 17 women (p=0.003) were of culturally and linguistically diverse background. Additionally, cervicitis was found to be significantly associated with TV, RR 1.66 (1.14 to 2.42), p=0.034. Conclusions Traditional TV-detection methods underestimate TV prevalence in urban Australia. The TV prevalence of 4.8% by PCR testing in this study exceeds previously reported urban Australian TV rates of <1%. An increase in trichomoniasis-associated adverse reproductive outcomes and enhanced HIV transmission poses a salient public health threat. Accordingly, TV warrants a higher profile in urban STI clinic settings in developed countries, and we suggest that priority be given to development of standardised molecular TV detection techniques and that these become part of routine STI testing.

Human cytomegalovirus directly modulates expression of chemokine CCL2 (MCP-1) during viral replication.

Human cytomegalovirus (CMV) infects monocytes and other haematopoietic progenitor cells which then act as reservoirs for latency and virus dissemination. The chemokine CCL2 (monocyte chemotactic protein-1 or MCP-1) exhibits potent chemotactic activity for monocytes and is a likely target for CMV-induced immunomodulation. In this study, we demonstrate CMV modulates CCL2 expression in MRC-5 fibroblasts with multiplicity-dependent kinetics, where CCL2 is upregulated during early stage infection, followed by CCL2 inhibition at late stage infection. This CMV-induced CCL2 modulation was dependent upon virus replication, as UV-inactivated virus did not elicit any changes in CCL2 levels. Dual immunofluorescence staining showed CMV strains AD169, purified AD169, Merlin, FIX WT (FLAG-US28/WT) and pUS28-deficient FIX (FIX-ΔUS28) all induced upregulation of CCL2 primarily within infected cells. Focal upregulation of CCL2 within FIX-ΔUS28-infected cells demonstrated intracellular CCL2 accumulation was independent of CCL2 sequestration by the CMV-encoded chemokine receptor US28. Infection with purified virus confirmed CMV-induced CCL2 upregulation was not due to any CCL2-inducing factors contained within non-purified virus stocks. The CMV-induced CCL2 expression kinetics occurred concurrently with modulation of the CCL2 transcriptional activators NF-κB, interferon regulatory factor 3 and cytokine IFN-β, independent of virus strain, and with the establishment of viral replication compartments within infected cell nuclei. This is the first report to our knowledge to demonstrate CMV modulation of CCL2 expression within infected cells during viral replication. This immune modulation may facilitate virus dissemination, establishment of latency and pathogenesis of CMV-induced host disease.

Diversity of antiviral-resistant human cytomegalovirus in heart and lung transplant recipients.

J.M. Iwasenko, G.M. Scott, Z. Naing, A.R. Glanville, W.D. Rawlinson. Diversity of antiviral‐resistant human cytomegalovirus in heart and lung transplant recipients.
Transpl Infect Dis 2011: 13: 145–153. All rights reserved

Viral factors influencing the outcome of human cytomegalovirus infection in liver transplant recipients

BACKGROUND Cytomegalovirus (CMV) remains the leading viral cause of disease following orthotopic liver transplantation (OLT) despite the availability of antiviral agents for prophylaxis and therapy. OBJECTIVE Examine the viral factors that influence the outcome of CMV infection following valganciclovir prophylaxis or laboratory-guided preemptive therapy in OLT recipients. STUDY DESIGN The value of valganciclovir prophylaxis and laboratory-guided preemptive therapy for the prevention of CMV infection and disease was observed in 64 OLT recipients. Prophylaxis was given to all CMV seronegative recipients receiving a liver from a seropositive donor (D+R-; n=15), and all other recipients were randomised to receive either prophylaxis (n=24) or laboratory-guided preemptive therapy (n=25). Recipients were monitored for CMV DNAemia, viral load, emergence of antiviral resistant strains and co-infections. RESULTS CMV end-organ disease and antiviral resistant strains only occurred in D+R- recipients despite the use of prophylaxis in these patients. The D+R- recipients commencing prophylaxis immediately following transplantation had better outcomes compared to those for whom prophylaxis was delayed due to renal impairment. Prophylaxis reduced the incidence of CMV DNAemia, persistent infection, and high viral loads for CMV seropositive (D-R+and D+R+) recipients, but laboratory-guided preemptive therapy effectively controlled CMV infection and prevented disease in these OLT recipients. CONCLUSION Delaying the commencement of valganciclovir prophylaxis may be associated with worse outcomes for high-risk OLT recipients. Laboratory-guided pre-emptive therapy remains an alternative approach for seropositive recipients at lower risk of CMV disease.