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1.
Microbiol Spectr ; 10(4): e0135822, 2022 08 31.
Article in English | MEDLINE | ID: mdl-35950846

ABSTRACT

High-throughput diagnostic assays are required for large-scale population testing for severe acute respiratory coronavirus 2 (SARS-CoV-2). The gold standard technique for SARS-CoV-2 detection in nasopharyngeal swab specimens is nucleic acid extraction followed by real-time reverse transcription-PCR. Two high-throughput commercial extraction and detection systems are used routinely in our laboratory: the Roche cobas SARS-CoV-2 assay (cobas) and the Roche MagNA Pure 96 system combined with the SpeeDx PlexPCR SARS-CoV-2 assay (Plex). As an alternative to more costly instrumentation, or tedious sample pooling to increase throughput, we developed a high-throughput extraction-free sample preparation method for naso-oropharyngeal swabs using the PlexPCR SARS-CoV-2 assay (Direct). A collection of SARS-CoV-2-positive (n = 185) and -negative (n = 354) naso-oropharyngeal swabs in transport medium were tested in parallel to compare Plex to Direct. The overall agreement comparing the qualitative outcomes was 99.3%. The mean cycle of quantification (Cq) increase and corresponding mean reduction in viral load for Direct ORF1ab and RdRp compared to Plex was 3.11 Cq (-0.91 log10 IU/mL) and 4.78 Cq (-1.35 log10 IU/mL), respectively. We also compared Direct to a four-sample pool by combining each positive sample (n = 185) with three SARS-CoV-2-negative samples extracted with MagNA Pure 96 and tested with the PlexPCR SARS-CoV-2 assay (Pool). Although less sensitive than Plex or Pool, the Direct method is a sufficiently sensitive and viable approach to increase our throughput by 12,032 results per day. Combining cobas, Plex, and Direct, an overall throughput of 19,364 results can be achieved in a 24-h period. IMPORTANCE Laboratories have experienced extraordinary demand globally for reagents, consumables, and instrumentation, while facing unprecedented testing demand needed for the diagnosis of SARS-CoV-2 infection. A major bottleneck in testing throughput is the purification of viral RNA. Extraction-based methods provide the greatest yield and purity of RNA for downstream PCR. However, these techniques are expensive, time-consuming, and depend on commercial availability of consumables. Extraction-free methods offer an accessible and cost-effective alternative for sample preparation. However, extraction-free methods often lack sensitivity compared to extraction-based methods. We describe a sensitive extraction-free protocol based on a simple purification step using a chelating resin, combined with proteinase K and thermal treatment. We compare the sensitivity qualitatively and quantitatively to a well-known commercial extraction-based system, using a PCR assay calibrated to the 1st WHO international standard for SARS-CoV-2 RNA. This method entails high throughput and is suitable for all laboratories, particularly in jurisdictions where access to instrumentation and reagents is problematic.


Subject(s)
COVID-19 Testing , COVID-19 , COVID-19/diagnosis , Humans , Nasopharynx , RNA, Viral/analysis , SARS-CoV-2/genetics , Specimen Handling/methods
2.
Diagn Microbiol Infect Dis ; 101(4): 115519, 2021 Dec.
Article in English | MEDLINE | ID: mdl-34571354

ABSTRACT

To improve laboratory safety we thermally treated naso-oropharyngeal samples before testing with the cobas SARS-CoV-2 assay. This study aimed to determine if thermal treatment significantly affects the qualitative detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the quantitative measurement of cobas SARS-CoV-2 ORF1a and E-gene target copy number using an in-house quantitative method. A collection of positive (n = 238) and negative samples (n = 196) was tested in parallel comparing thermal treatment (75 °C for 15 minutes) to room-temperature. There were no significant differences in the final qualitative outcomes for thermal treatment versus room-temperature (99.8% agreement) despite a statistically significant reduction (P < 0.05) in target copy number following thermal treatment. The median ORF1a and E-gene reduction in target copy number was -0.07 (1.6%) and -0.22 (4.2%) log10 copies/mL respectively. The standard curves for both ORF1a and E-gene targets were highly linear (r2 = 0.99). Good correlation was observed for ORF1a (r2 = 0.96) and E-gene (r2 = 0.98) comparing thermal treatment to room-temperature control.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Nasopharynx/virology , Oropharynx/virology , SARS-CoV-2/isolation & purification , Specimen Handling/methods , Hot Temperature , Humans , RNA, Viral/isolation & purification , Virus Inactivation
4.
Pathology ; 50(5): 554-561, 2018 Aug.
Article in English | MEDLINE | ID: mdl-29945729

ABSTRACT

Prevention of cytomegalovirus (CMV) infection remains an important aspect of improving long term outcomes of solid organ transplantation and currently relies on prophylactic antiviral medication and early detection of viraemia or disease. Uptake of diagnostic tools to personalise assessment of CMV immunity and guide interpretation of viral testing remains low. We assessed the QuantiFERON-CMV assay in 54 Western Australian recipients of renal, heart, lung or liver allografts to determine the relationship between CMV-specific immunity, viraemia and disease following cessation of antiviral prophylaxis. We carried out an initial validation study which demonstrated that the QuantiFERON-CMV assay is highly precise and strongly correlated with CMV-specific antibodies in 30 healthy blood donors (sensitivity 82%, specificity 95%). In the solid organ transplant recipients we examined, the prevalence of asymptomatic CMV viraemia was high at 61% but only two patients ultimately developed CMV disease, both of whom had negative QuantiFERON-CMV responses, indicating lack of CMV T-cell immunity. The vast majority (94%) of patients who had spontaneous resolution or stability of asymptomatic CMV viraemia without any antiviral treatment had positive QuantiFERON-CMV responses. Positive QuantiFERON-CMV responses at cessation of antiviral prophylaxis were significantly associated with pre-transplant CMV seropositivity and the development of asymptomatic viraemia post-transplantation. Overall, 27% of patients were recommenced on antiviral therapy because of asymptomatic CMV viraemia. Patients with non-reactive QuantiFERON-CMV responses had earlier onset, higher level CMV viraemia compared to those with positive QuantiFERON-CMV responses, although the difference did not reach statistical significance. QuantiFERON-CMV results may contribute to decision making in concert with the serological risk profile, net state of immunosuppression and CMV viral load.


Subject(s)
Antiviral Agents/pharmacology , Cytomegalovirus Infections/diagnosis , Cytomegalovirus Infections/drug therapy , Immunocompromised Host/drug effects , Antibodies, Viral/therapeutic use , CD8-Positive T-Lymphocytes/immunology , Cytomegalovirus Infections/immunology , Female , Humans , Kidney Transplantation/methods , Male , Viremia , Western Australia
5.
Papillomavirus Res ; 4: 90-98, 2017 12.
Article in English | MEDLINE | ID: mdl-29179876

ABSTRACT

BACKGROUND: Anal intraepithelial neoplasia is associated with high-risk human papillomavirus (hrHPV) as a precursor to anal cancer. However, factors other than hrHPV are likely to be involved and further study of cofactors is required because of the possibility of syndemic interactions. METHODS: Three hundred and fourteen patients underwent 457 operations. Histopathology and hrHPV testing using the Digene Hybrid Capture 2 (HC 2) method were performed. Demographic factors and sexually transmissible infections (STIs) were recorded. RESULTS: Results showed that hrHPV alone was associated with HSIL (OR = 4.65, p < 0.001). None of the other STIs were alone associated with HSIL but amplification of risk was found when hrHPV infection occurred with HIV (OR = 11.1); syphilis (OR = 5.58); HSV 2 (OR = 7.85); gonorrhoea (OR = 6.45) and some other infections. CONCLUSIONS: These results suggest that hrHPV is a sufficient cause of anal HSIL. Seropositivity for HIV, HSV 2, T. pallidum, HBV and HCV and a history of gonorrhoea or chlamydia exert a powerful amplifying factor increasing the risk of HSIL above the risk with hrHPV alone. Other co-factors which are associated with an increased risk of HSIL are increased age, male gender, MSM behaviour and self-reported history of more than 50 sexual partners. This pattern of disease in patients with warts is characteristic of a syndemic with potential serious increased risk of anal carcinoma.


Subject(s)
Anus Neoplasms/virology , Papillomavirus Infections/complications , Papillomavirus Infections/virology , Sexually Transmitted Diseases/complications , Squamous Intraepithelial Lesions of the Cervix/virology , Adolescent , Adult , Age Factors , Aged , Anal Canal/pathology , Anus Neoplasms/etiology , Anus Neoplasms/physiopathology , Carcinoma in Situ/complications , Carcinoma in Situ/virology , Female , Homosexuality, Male , Humans , Male , Middle Aged , Papanicolaou Test , Papillomaviridae/isolation & purification , Sex Factors , Sexual Behavior , Sexual and Gender Minorities , Sexually Transmitted Diseases/epidemiology , Sexually Transmitted Diseases/microbiology , Sexually Transmitted Diseases/virology , Uterine Cervical Neoplasms/complications , Uterine Cervical Neoplasms/virology , Young Adult , Uterine Cervical Dysplasia/complications
6.
Clin Immunol ; 179: 54-63, 2017 06.
Article in English | MEDLINE | ID: mdl-28315414

ABSTRACT

Antibody responses have not been fully characterised in chronically HIV/HCV patients receiving antiretroviral therapy (ART). Seventeen HIV/HCV patients receiving ART were followed for a median (range) interval of 597 (186-766) weeks. Prior to ART, HIV/HCV patients had lower levels of antibodies reactive with HCV core and JFH-1, and lower genotype cross-reactive neutralising antibodies (nAb) titres, than HCV patients. Levels of JFH-1 reactive antibody increased on ART, irrespective of CD4+ T-cell counts or changes in serum ALT levels. The appearance of nAb coincided with control of HCV viral replication in five HIV/HCV patients. In other patients, HCV viral loads remained elevated despite nAb responses. Sustained virological responses following HCV therapy were associated with reduced antibody responses to JFH-1 and core but elevated responses to non-structural proteins. We conclude that nAb responses alone may fail to clear HCV, but contribute to control of viral replication in some HIV/HCV patients responding to ART.


Subject(s)
Antibodies, Neutralizing/blood , Antiviral Agents/therapeutic use , HIV Infections/immunology , Hepatitis C/immunology , Adult , Antibodies, Viral/blood , Antiviral Agents/pharmacology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Line, Tumor , Female , Genotype , HIV/genetics , HIV Infections/drug therapy , HIV Infections/virology , Hepacivirus/genetics , Hepacivirus/immunology , Hepatitis C/drug therapy , Hepatitis C/virology , Humans , Immunoglobulin G/blood , Lymphocyte Count , Male , RNA, Viral/blood , Viral Load
7.
BMC Infect Dis ; 16: 342, 2016 07 22.
Article in English | MEDLINE | ID: mdl-27448566

ABSTRACT

BACKGROUND: BK virus is a polyoma virus causing renal allograft nephropathy. Reduction of immunosuppression with the early recognition of significant BK viral loads in urine and plasma can effectively prevent BKV associated nephropathy (BKVN), however the optimal compartment and frequency of BK viral load measurement post renal transplantation are undetermined. Our purpose was to examine time to detection and viral loads in urine compared to plasma, and establish viral load cut-offs associated with histological BKVN. METHODS: We performed a retrospective analysis of the BKV screening frequency and compartment(s) of 277 adult renal transplant recipients (RTR). RESULTS: BKVN was histologically diagnosed in 17 (6.1 %) RTR. In cases where both urine and plasma were tested fortnightly for 6 months (n = 53), BKV was detected in the urine 29 days earlier than plasma. Fortnightly (n = 72) versus 3-monthly (n = 78) testing demonstrated that BKV was detected in the urine significantly earlier (median 63 versus 97 days, p = 0.001) and at a lower level (median 3.27 versus 6.71 log10 c/mL, p < 0.001) with more frequent testing, but this difference was not evident in plasma first detection (80 versus 95 days, p = 0.536) or first positive viral load (3.18 versus 3.30 log10 c/mL, p = 0.603). The optimum cut-off BK viral load for histological diagnosis of BKVN was 4.10 log10 c/mL for the first positive urine, 3.79 log10 c/mL for the first positive plasma, 9.24 log10 c/mL for the peak urine, and 4.53 log10 c/mL for the peak plasma. CONCLUSIONS: Frequent urinary BK viral load screening for the prevention of BKVN is suggested due to its high sensitivity and earlier detection.


Subject(s)
BK Virus/isolation & purification , DNA, Viral/blood , DNA, Viral/urine , Kidney Diseases/diagnosis , Polyomavirus Infections/diagnosis , Tumor Virus Infections/diagnosis , Adult , BK Virus/growth & development , DNA, Viral/analysis , Early Diagnosis , Female , Humans , Kidney Diseases/blood , Kidney Diseases/urine , Kidney Diseases/virology , Kidney Transplantation/adverse effects , Male , Middle Aged , Polyomavirus Infections/blood , Polyomavirus Infections/complications , Polyomavirus Infections/urine , Prognosis , Retrospective Studies , Serologic Tests , Transplant Recipients , Transplantation, Homologous/adverse effects , Tumor Virus Infections/blood , Tumor Virus Infections/complications , Tumor Virus Infections/urine , Viral Load/methods
8.
J Gastroenterol Hepatol ; 30 Suppl 2: 1-5, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25641223

ABSTRACT

The evolution of management of hepatitis C virus (HCV) has seen a majority of patients treated being regarded as cured. Despite this development, uptake of treatment remains low in Australia, and this is particularly true in rural and remote areas. The largest state in Australia, Western Australia (WA), covers an area of 2500 km(2). As the rural and remote population of WA is scattered in small areas rather than major centers, poor accessibility to remote areas and lack of adequate of medical and nursing resources pose major problems in providing equity of care to patients with chronic HCV. A statewide hepatitis model of care, established in 2009, has led to an increase in identification and treatment of patients living with HCV. Strategies used to facilitate these changes include telehealth, a nurse practitioner model, and general practitioner shared-care model. The statewide program will be modified to meet the changing needs of patients as all-oral treatment regimens become available, with further emphasis being placed on the role of rural and remote health professionals in identifying patients with HCV and initiating and monitoring treatment.


Subject(s)
Delivery of Health Care, Integrated , Health Services Accessibility/statistics & numerical data , Hepatitis C, Chronic/epidemiology , Hepatitis C, Chronic/therapy , Rural Health Services/statistics & numerical data , Rural Health Services/trends , Delivery of Health Care, Integrated/statistics & numerical data , Delivery of Health Care, Integrated/trends , Health Resources/supply & distribution , Humans , Quality of Health Care/statistics & numerical data , Quality of Health Care/trends , Remote Consultation , Rural Population/statistics & numerical data , Western Australia/epidemiology
9.
J Clin Microbiol ; 52(8): 2829-33, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24871216

ABSTRACT

Active surveillance is part of a multifaceted approach used to prevent the spread of vancomycin-resistant enterococci (VRE). The impact of fecal density, the vancomycin MIC of the isolate, and the vancomycin concentration in liquid medium on test performance are uncertain. Using fecal specimens spiked with a collection of 18 VRE (predominantly vanB) with a wide vancomycin MIC range, we compared the performances of commercial chromogenic agars (CHROMagar VRE, chromID VRE, Brilliance VRE, and VRE Select) and 1 liquid medium (Enterococcosel enrichment broth) for VRE detection. The specificity of solid media was excellent; however, the sensitivity at 48 h varied from 78 to 94%. Screening using liquid medium was less sensitive than screening with solid media, particularly as the vancomycin content increased. Sensitivity declined (i) as the fecal VRE density decreased, (ii) when the media were assessed at 24 h (versus 48 h), and (iii) for isolates with a low vancomycin MIC (sensitivity, 25 to 75% versus 100% for isolates with vancomycin MIC of <16 mg/liter versus >32 mg/liter on solid medium using 10(6) CFU/ml of feces). Depending on local epidemiology and in particular VRE vancomycin MICs, the sensitivity of culture-based methods for VRE screening of stool or rectal specimens may be suboptimal, potentially facilitating secondary transmission.


Subject(s)
Anti-Bacterial Agents/pharmacology , Enterococcus/drug effects , Feces/microbiology , Vancomycin Resistance , Vancomycin/pharmacology , Culture Media/chemistry , Enterococcus/isolation & purification , Gram-Positive Bacterial Infections/microbiology , Humans , Microbial Sensitivity Tests/methods , Sensitivity and Specificity
10.
Am J Trop Med Hyg ; 88(3): 481-9, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23296449

ABSTRACT

A review of the laboratory-confirmed cases of Murray Valley encephalitis (MVE) from Western Australia between 2009 and 2011 was conducted to describe the clinical, laboratory, and radiological features of the disease. The nine encephalitis patients presented with altered mental state and seizures, tremor, weakness, or paralysis. All patients developed a raised C-reactive protein, whereas most developed acute liver injury, neutrophilia, and thrombocytosis. All patients with encephalitis developed cerebral peduncle involvement on early magnetic resonance imaging (MRI). The absence of thalamic MRI hyperintensity during the acute illness, with or without leptomeningeal enhancement, predicted a better neurological outcome, whereas those patients with widespread abnormalities involving the thalamus, midbrain, and cerebral cortex or the cerebellum had devastating neurological outcomes. MRI scans repeated months after acute illness showed destruction of the thalamus and basal ganglia, cortex, or cerebellum. These findings may help clinicians predict the neurological outcome when evaluating patients with MVE.


Subject(s)
Brain/pathology , Encephalitis Virus, Murray Valley , Encephalitis, Arbovirus/diagnostic imaging , Encephalitis, Arbovirus/pathology , Adult , Aged , Child, Preschool , Encephalitis, Arbovirus/epidemiology , Female , Humans , Male , Middle Aged , Radiography , Retrospective Studies , Treatment Outcome , Western Australia/epidemiology
11.
Emerg Med Australas ; 23(4): 502-6, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21824318

ABSTRACT

OBJECTIVE: The aim of the present study was to determine if the quantification of bacterial 16S rDNA could be clinically useful in predicting patients at increased risk of developing septic shock. METHODS: A retrospective study of patients with positive blood cultures taken on arrival to the ED. An EDTA sample was collected simultaneously with blood cultures and assayed by polymerase chain reaction to quantitate the bacterial 16S rDNA load. Descriptive and clinical data were collected from the medical record and this was blinded to the 16S rDNA result. Subsequently, the 16S rDNA result was compared with illness severity markers including septic shock and death to determine the relationship between the 16S rDNA load and illness severity. RESULTS: 98 patients (mean age 61 ± 20 years, range 18-92) with positive blood cultures were studied, most commonly growing Escherichia coli (n= 25) and Staphylococcus aureus (n= 23). 16 (16%) died. There were 42 (43%) 16S rDNA positive patients. A high 16S rDNA load was associated with an increased risk of developing delayed septic shock (OR 21.9, 95% CI 2.5-192.6) in comparison with either a low or negative 16S rDNA load; with a mortality OR 4.6 (95% CI 0.9-23.5). CONCLUSIONS: The quantitative assay for 16S rDNA might be a useful screening tool to detect severe sepsis in those whom it might not be clinically suspected. However, prospective studies are required to further assess the clinical usefulness of this assay.


Subject(s)
DNA, Bacterial/analysis , RNA, Ribosomal, 16S/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Sepsis/diagnosis , Adolescent , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Retrospective Studies , Sepsis/microbiology , Sepsis/mortality , Shock, Septic/prevention & control , Young Adult
12.
J Infect Dis ; 204(3): 400-7, 2011 Aug 01.
Article in English | MEDLINE | ID: mdl-21742838

ABSTRACT

On 16 April 2009, a boat carrying 47 Afghan asylum seekers and 2 Indonesian crew exploded in Australian waters, resulting in mass casualties. Of these casualties, 23 persons who suffered significant burns were transferred to Royal Perth Hospital, Perth, Western Australia. One patient was subsequently shown to be a hepatitis B virus (HBV) carrier at the time of the explosion. Over the following months, 3 other patients received a diagnosis of acute hepatitis B, and an additional 4 patients showed serological evidence of recent HBV infection, including 1 patient who was transferred to another Australian city. Molecular typing determined that the strains from the HBV carrier and the acute and recent case patients formed a closely related cluster, and the investigation suggested that transmission occurred at or around the time of the boat explosion. This is the first report of confirmed transmission of HBV following a disaster, and it reinforces the importance of postexposure prophylaxis for HBV in mass casualty situations.


Subject(s)
Disease Outbreaks , Hepatitis B/epidemiology , Mass Casualty Incidents , Refugees , Adolescent , Adult , Afghanistan , Australia/epidemiology , Genotype , Hepatitis B/transmission , Hepatitis B Surface Antigens/blood , Hepatitis B virus/classification , Hepatitis B virus/genetics , Humans , Male , Middle Aged , Phylogeny
13.
Dis Markers ; 28(5): 273-80, 2010.
Article in English | MEDLINE | ID: mdl-20592450

ABSTRACT

The role of pro-fibrogenic cytokines in the outcome of infections with hepatitis C virus (HCV) and the response to treatment with pegylated interferon-alpha (pegIFNalpha) and ribavirin remains unclear. To address this issue, we assessed hepatic fibrosis and plasma markers pertinent to T-cell mediated fibrogenesis and inflammation at the start of treatment. Levels of soluble (s)CD30, interleukin-13 receptor alpha 2 (IL-13Ralpha2), total and active transforming growth factor-beta 1 (TGFbeta1), interleukin-18 (IL-18) and interferon-gamma inducible protein-10 (IP-10, CXCL10) were correlated with the severity of fibrosis and with treatment outcome using multiple logistic regression modelling. The Hepascore algorithm was confirmed as a marker of fibrosis, but was a poor predictor of treatment outcome. Inclusion of all immunological markers improved prediction based on Hepascore alone (p=0.045), but optimal prediction was achieved with an algorithm ("TIPscore") based on TGFbeta1 (total), IP-10, age, sex and HCV genotype (p=0.003 relative to Hepascore). Whilst this was only marginally more effective than predictions based on HCV genotype age and sex (p=0.07), it associates high TGFbeta1 and low IP-10 levels with a failure of therapy.


Subject(s)
Antiviral Agents/therapeutic use , Chemokine CXCL10/blood , Extracellular Matrix Proteins/blood , Hepacivirus/physiology , Hepatitis C/drug therapy , Transforming Growth Factor beta/blood , Viral Load , Adult , Aged , Algorithms , Area Under Curve , Female , Hepatitis C/blood , Hepatitis C/virology , Humans , Interferon alpha-2 , Interferon-alpha/therapeutic use , Liver Cirrhosis/diagnosis , Liver Cirrhosis/drug therapy , Male , Middle Aged , Polyethylene Glycols/therapeutic use , Recombinant Proteins , Ribavirin/therapeutic use , Sensitivity and Specificity , Treatment Outcome
14.
Cell Immunol ; 264(2): 150-5, 2010.
Article in English | MEDLINE | ID: mdl-20579979

ABSTRACT

Toll-like receptor (TLR) expression on T-cells and the signalling pathways that lead to the production of cytokines may limit antigen-specific T-cell responses. Here, expression of TLR and retinoic acid inducible gene I (RIG-I) on T-cells were evaluated in patients chronically infected with hepatitis C virus (HCV), before and during pegylated interferon-alpha and ribavirin therapy. Expression of TLR2,3,4,7,9 and retinoic acid inducible gene (RIG)-I on different CD4(+) and CD8(+) T-cell sub-populations (naïve: CD45RA(+)CD57(-); central memory: T(CM) CD45RA(-)CD57(-); effector memory: T(EM) CD45RA(-)CD57(+) and terminally differentiated effector memory: T(EMRA) CD45RA(+)CD57(+)) were measured by flow cytometry. TLR7, TLR9 and RIG-I expression on CD4(+) T-cells and RIG-I expression on CD8(+) T-cells was higher in patients than healthy controls. Therapy increased expression of TLR2, TLR4 and TLR9 and this was observed for all T-cell sub-populations. Evaluation of TLR expression at baseline did not identify patients able to achieve sustained virological response following therapy.


Subject(s)
CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Hepacivirus/immunology , Hepatitis C, Chronic/immunology , Toll-Like Receptors/metabolism , Adult , Aged , Antigens, CD/biosynthesis , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Cell Separation , DEAD Box Protein 58 , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/immunology , DEAD-box RNA Helicases/metabolism , Female , Flow Cytometry , Hepacivirus/pathogenicity , Hepatitis C, Chronic/drug therapy , Hepatitis C, Chronic/pathology , Hepatitis C, Chronic/physiopathology , Humans , Immunologic Memory , Interferon alpha-2 , Interferon-alpha/administration & dosage , Male , Middle Aged , Polyethylene Glycols/administration & dosage , Receptors, Immunologic , Recombinant Proteins , Ribavirin/administration & dosage , Toll-Like Receptors/genetics , Toll-Like Receptors/immunology
15.
J Med Virol ; 82(4): 568-74, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20166183

ABSTRACT

Natural killer (NK) cells are implicated in the regulation of a protective immune response in patients chronically infected with hepatitis C virus (HCV), but effects of interferon-alpha/ribavirin therapy on NK cell subsets and the consequences of viral clearance during therapy remain unclear. Samples were collected from chronically infected patients (n = 34) at baseline and from a subset after 3-10 months on pegylated interferon-alpha and ribavirin therapy (n = 19). NK cells present in cryopreserved PBMC were characterized by flow cytometry. Before therapy, the frequency of CD3-CD56+ NK cells was lower in patients than uninfected controls. Therapy increased proportions of CD56(bright) NK cells. Frequencies of CD56(dim) NK cells declined slightly while perforin and CD16 expression on CD56(dim) NK cells decreased compared to baseline samples. Evaluation of NK cell subsets at baseline did not identify patients able to achieve sustained virological response following therapy. However, therapy may promote the expansion of NK cells able to produce interferon-gamma, while minimizing cytotoxicity to limit liver damage.


Subject(s)
Antiviral Agents/therapeutic use , CD56 Antigen/analysis , Hepatitis C, Chronic/drug therapy , Hepatitis C, Chronic/immunology , Interferon-alpha/therapeutic use , Killer Cells, Natural/immunology , Ribavirin/therapeutic use , Adult , Aged , CD3 Complex/analysis , Female , Flow Cytometry , Humans , Killer Cells, Natural/chemistry , Lymphocyte Subsets/chemistry , Lymphocyte Subsets/immunology , Male , Middle Aged , Young Adult
17.
J Gastroenterol Hepatol ; 24(6): 1017-23, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19054259

ABSTRACT

BACKGROUND: Treatment of chronic hepatitis C with interferon is known to be associated with thyroid dysfunction (TD) in 5-14% of patients. We studied the incidence, types, outcome and risk factors predictive of thyroid dysfunction. METHODS: A retrospective analysis was performed on all patients treated with interferon alpha (IFN) or pegylated interferon alpha (PEG-IFN) +/- ribavirin (RBV), who developed abnormal thyroid function tests (TFTs). These cases were compared with treatment-matched controls to identify factors predictive of thyroid dysfunction. Statistical methods consisted of: chi(2) test, Fischer's exact test, Welch's t-test, and multivariate analysis. RESULTS: From a total of 511 patients, 45 cases with TD were identified (8.8%). Pegylated interferon alpha was associated with higher rates of TD than IFN (14.1% vs 6.0%, P = 0.0029). Female sex (OR 5.6, 95% CI 1.1-7) and Asian ethnicity (OR 2.7, 95% CI 1.4-22) were independent predictors of developing TD. Cytology was obtained in 13 patients: benign follicular pattern (8); thyroiditis (3); and normal (2). Thyroid peroxidase (TPO) antibodies (P = 0.004) and earlier onset of dysfunction (P = 0.03) were associated with need for treatment. Sixteen patients had persistent TD by the end of the follow-up period, predicted by female sex, non-Asian ethnicity, prior history of TD and TPO antibodies. CONCLUSIONS: Pegylated interferon alpha, female sex and Asian ethnicity are independent risk factors for TD. Thyroid peroxidase antibodies and earlier TD within the course of IFN are associated with the requirement for treatment. Thyroid function tests should be monitored during and after IFN-based therapy. The most common cytological finding is a benign follicular pattern.


Subject(s)
Hepatitis C, Chronic/drug therapy , Interferon-alpha/adverse effects , Thyroid Diseases/chemically induced , Adult , Case-Control Studies , Chi-Square Distribution , Drug Therapy, Combination , Female , Humans , Interferon alpha-2 , Interferon-alpha/therapeutic use , Male , Middle Aged , Recombinant Proteins , Retrospective Studies , Ribavirin/adverse effects , Ribavirin/therapeutic use , Risk Factors , Sex Factors , Thyroid Function Tests , Western Australia
18.
Diagn Microbiol Infect Dis ; 61(3): 302-8, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18378109

ABSTRACT

BK virus (BKV) may cause nephropathy in renal transplant patients, resulting in graft dysfunction and possible graft loss. We used a sensitive quantitative BKV assay to monitor plasma BK viral loads in 11 renal transplant patients for periods ranging from 37 to 189 weeks posttransplant. Five patients remained negative for BKV, and 6 developed viremia, including 1 patient with a transient viremia. Of the viremic patients, 2 were diagnosed with BKV nephropathy after increasing serial BK viral loads, prompting a renal biopsy that established the diagnosis. A 3rd patient had high initial BK viral load and biopsy-proven disease that resolved with reduced immunosuppression. Two patients did not develop nephropathy despite persistent viral loads of 10(4) copies/mL. Five of 6 patients experienced viral clearance from the plasma (BK viral load <500 copies/mL), which was associated with their renal function becoming stabilized, and the remaining patient experienced a downward trend in viral load and stable renal function. Thus, the BKV quantitative assay was useful in aiding the diagnosis of BKV nephropathy, monitoring the response to reductions in immunosuppression and identified that some patients can have persistent viremia and still develop stable renal function without specific antiviral therapy.


Subject(s)
BK Virus/isolation & purification , Kidney Transplantation , Polyomavirus Infections/virology , Tumor Virus Infections/virology , Viral Load , Adult , Female , Humans , Longitudinal Studies , Male , Middle Aged
19.
Immunol Cell Biol ; 84(4): 390-5, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16834574

ABSTRACT

Rapid progression of hepatitis C virus (HCV) disease in patients with HIV/HCV may reflect different cytokine responses and be influenced by HCV genotype. This is addressed by a study of patients with HIV/HCV coinfection and infection with HCV genotype 2 or 3 (2/3). They are compared with coinfected patients infected with genotype 1 and HCV monoinfected patients matched for HCV genotype. IFN-gamma, IL-10, IL-4 and IL-4delta2 mRNA were quantified by real-time PCR in unstimulated PBMC and after in vitro stimulation with HCV core or nonstructural 3/4A antigen. In unstimulated PBMC, levels of IFN-gamma and IL-4 mRNA were lowest in HIV/HCV genotype 1 patients, intermediate in HIV/HCV genotype 2/3 patients and highest in HCV genotype 2/3 patients. Neither HCV genotype nor HIV affected levels of IL-10 mRNA in unstimulated PBMC or IFN-gamma, IL-4 and IL-10 mRNA in PBMC stimulated with HCV antigens. Levels of IL-4 and IL-4delta2 mRNA correlated in mitogen-stimulated PBMC from all patient groups but both were low in HIV/HCV genotype 1 patients. Serum soluble CD30 levels (a putative marker of a T2 cytokine environment) did not differ between patient groups. The data do not suggest a shift in the T1/T2 balance driven by HIV coinfection or HCV genotype but either may affect IL-4 bioavailability.


Subject(s)
Cytokines/genetics , HIV Infections/immunology , HIV/immunology , Hepacivirus/genetics , Hepatitis C/immunology , RNA, Messenger/metabolism , Th1 Cells/immunology , Th2 Cells/immunology , Adult , Cytokines/biosynthesis , Cytokines/immunology , Genotype , HIV Infections/virology , Hepacivirus/immunology , Hepatitis C/genetics , Hepatitis C/virology , Hepatitis C Antigens/immunology , Humans , Interferon-gamma/immunology , Interleukin-4/immunology , Leukocytes, Mononuclear/immunology , Middle Aged , RNA, Messenger/genetics
20.
J Gastroenterol Hepatol ; 21(7): 1184-8, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16824073

ABSTRACT

BACKGROUND AND AIM: Hepatitis C virus (HCV) is a common infection with serious health consequences. Alternative therapies are often used for hepatitis C. The aim of the present study was to examine CH100, a Chinese herbal remedy, for efficacy in therapy of chronic HCV. METHODS: A randomized double blind placebo-controlled study in a tertiary outpatient clinic of CH100 over 24 weeks with 24 weeks follow-up in patients with chronic HCV infection. Alanine aminotransferase (ALT), HCV-RNA, quality of life (by SF-36) and side-effects were examined regularly. Ninety-seven patients were enrolled of which 91 were suitable for analysis. RESULTS: No significant differences were observed between patients receiving CH100 (n = 61) or placebo (n = 30) at baseline or during follow-up in either ALT or viral titer. However, patients receiving CH100 had a fall in mean ALT over time (P = 0.05 at week 4, P = 0.26 at week 12, and P = 0.04 at week 24), with reversion to baseline during post-treatment follow up. No significant side-effects were observed although mild complaints were common. Quality of life scores improved in both groups with time, and bodily pain significantly improved in CH100 recipients. CONCLUSION: CH100 appears to be no better than placebo in the treatment of patients with chronic HCV infection.


Subject(s)
Drugs, Chinese Herbal/therapeutic use , Hepatitis C, Chronic/drug therapy , Adult , Alanine Transaminase/blood , Double-Blind Method , Female , Follow-Up Studies , Hepacivirus/drug effects , Hepacivirus/genetics , Hepatitis C, Chronic/enzymology , Hepatitis C, Chronic/virology , Humans , Male , Polymerase Chain Reaction , Quality of Life , RNA, Viral/analysis , Retrospective Studies , Treatment Outcome , Viral Load
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