James P. Flexman

Hepatitis C virus-associated hepatitis following treatment of HIV-infected patients with HIV protease inhibitors: an immune restoration disease?

Objective:To report observations from case studies on the pathogenic mechanisms underlying the acute hepatitis that sometimes occurs in hepatitis C virus (HCV) and HIV coinfected patients following treatment with potent antiretroviral therapy that includes a HIV protease inhibitor. Methods:Cases of acute hepatitis were identified from a group of 133 patients enrolled in a retrospective study of pathogen-associated inflammatory disease following the use of potent antiretroviral therapy. Data on serum alanine aminotransferase concentrations, clinical events, HCV antibodies, and liver biopsies were collected from medical records. HCV RNA assays and additional HCV antibody assays were undertaken on stored plasma or sera. Results:Three of the 133 patients (2%) developed symptomatic hepatitis. One was HCV antibody-positive prior to commencing antiretroviral therapy and developed hepatitis subsequent to an episode of Mycobacterium avium complex disease associated with immune restoration. However, the other two patients had previously undiagnosed HCV infection for up to 2 years prior to antiretroviral therapy, with HCV RNA detected but anti-HCV antibody repeatedly undetectable in stored plasma or sera. HCV antibody was only detectable after antiretroviral therapy-induced decrease in plasma HIV RNA and immunological reconstitution. Plasma HCV RNA increased after therapy in one of these patients, but in the other the level was not increased at a time of active hepatitis demonstrated by liver biopsy. Conclusions:Hepatitis in HCV–HIV-coinfected patients following treatment with potent antiretroviral therapy may reflect restoration of anti-HCV immune responses rather than increased HCV replication or a hepatotoxic effect of antiretroviral therapy.

Rapid Detection of vanA and vanB Genes Directly from Clinical Specimens and Enrichment Broths by Real-Time Multiplex PCR Assay

ABSTRACT A real-time PCR assay previously developed for use on the Roche LightCycler platform was investigated as an alternative to culture for the direct detection of vancomycin-resistant enterococci (VRE) in clinical specimens. PCR primers and fluorescence resonance energy transfer hybridization probes specific for the vanA and vanB genes were combined in a multiplex real-time PCR assay performed directly with fecal material obtained by rectal swabbing and with enrichment broth samples. DNA was prepared from the rectal swabs and enrichment broths with a commercially available DNA preparation column designed specifically for use with fecal specimens. One hundred eighty duplicate rectal swabs were obtained from 42 patients who were previously found to be positive for VRE and who were being monitored for carriage of VRE. Direct and enrichment broth cultures were performed with one swab, while PCR was performed with the other swab as well as any corresponding presumptive positive enrichment broth. In total, 100 specimens from 30 patients remained positive for VRE by at least one method. The multiplex real-time PCR was positive for 88 enrichment broths of rectal swabs from 27 patients but for only 45 rectal swabs from 15 patients. Direct culture was positive for VRE for only 43 specimens from 11 patients, while enrichment broth culture was positive for VRE for 75 specimens from 22 patients. Inhibition studies for the multiplex real-time PCR assay, performed by spiking the DNA extracts from 50 negative rectal swabs and the corresponding enrichment broths with between 1 and 10 CFU of a VanB Enterococcus faecium strain, detected inhibition rates of 55.1 and 10%, respectively. PCR performed directly with enrichment broths was found to be significantly more sensitive than enrichment broth culture (P < 0.025). Negative samples were identified significantly earlier by PCR than by culture alone.

Real-time PCR for the rapid detection of vanA and vanB genes

A real-time PCR assay suitable for use on the Roche LightCycler platform was developed to replace an existing gel-based PCR assay for the simultaneous detection of the vanA & vanB genes in enterococcal isolates. Novel Fluorescence Resonance Energy Transfer (FRET) hybridization probes were designed. The multiplex real-time PCR assay and the existing gel-based assay were 100% concordant and both correctly detected the vanA or vanB genes in 4/4 VanA E. faecium and 25/25 VanB E. faecium. Additionally, 1/1 VanC1 E. gallinarum, 1/1 VanC2 E. casseliflavus and 47/47 vancomycin susceptible enterococci were negative for the vanA and vanB genes in both PCR assays. Results were available within 1.5 h for the real-time PCR assay compared to up to 5.5 h for the conventional PCR assay.

Bartonella henselae is a causative agent of cat scratch disease in Australia

We report the first isolation of Bartonella henselae from the blood and fleas of a cat of a patient with cat scratch disease (CSD) in Australia. A 49-year-old man presented with a history that 3 weeks after he had removed fleas from his cat he had developed fever, lethargy and anorexia for 3 days. This was followed by the appearance of axillary lymphadenopathy. There was no history of a bite or scratch and no primary lesion on the skin. Two fine needle aspirates of the axillary lymph node showed granulomatous lymphadenitis with no organisms seen by Warthin-Starry silver staining or electron microscopy. No organism was cultured from the patients lymph node aspirates or blood cultures processed by lysis centrifugation. However, the polymerase chain reaction (PCR) using p24E and p12B primers gave a 280 bp band indistinguishable from Bartonella henselae when using DNA extracted from the lymph node aspirates and the patients blood leucocytes. DNA sequencing of the PCR product from the patients blood showed that the DNA was from Bartonella henselae. The patients serum had a titre of 1024 in an indirect immunofluorescence antibody test for Bartonella henselae. Bartonella henselae was subsequently cultured from fleas and blood taken from the patients cat. This case provides evidence that Bartonella henselae is a causative agent of CSD in Australia and supports a possible role for fleas in transmission of the disease.

The frequency of high-grade intraepithelial neoplasia in anal/perianal warts is higher than previously recognized

A retrospective review of the prevalence of intraepithelial neoplasia (IN) in surgically removed perianal/anal warts from December 1995 to December 2004 was undertaken in patients referred to the Sexual Health Clinic at Royal Perth Hospital. Data were analysed from 115 men and 38 women, 29 of whom had HIV infection (27 men and two women). Perianal/anal IN within the warts was found in 78% (52% high grade) of men with HIV infection. In men without HIV infection, the overall rate of IN within warts was 33% (20% high grade). The IN rate was 8.3% for HIV-negative women (2.8% high grade). Rates of IN within perianal/anal warts in men with or without HIV infection are higher than previously reported, and suggest the likelihood of a substantial increase in the future incidence of anal cancer. The association between IN and genital warts needs to be further studied.

Use of real-time PCR and the LightCycler system for the rapid detection of Pneumocystis carinii in respiratory specimens

Pneumocystis carinii pneumonia (PCP) remains a major cause of morbidity and mortality in immunocompromised patients, including those infected with human immunodeficiency virus (HIV). The advent of real-time PCR technology offers the potential for rapid PCR results for the detection of P. carinii. In this report we describe the modification and evaluation of an existing PCR-based method for the detection of P. carinii DNA, into a real-time PCR assay suitable for use with the LightCycler system. Twenty eight induced sputum and bronchial washing specimens from 28 patients were tested by both a conventional PCR assay and a real-time PCR assay. Twelve specimens (42.9%) were positive in both the conventional and real-time PCR assays and sixteen (57.1%) were negative in both assays. The melting points of the amplified P. carinii DNA product obtained by melting curve analysis by the LightCycler of all P. carinii positive specimens ranged from 81.5 degrees C to 83.9 degrees C. There were no discordant results between the two assays for any of the specimens tested and results were available within 2 h for the real-time PCR assay compared to up to 11 h for the conventional PCR assay.

Toll-like receptor (TLR) expression on CD4+ and CD8+ T-cells in patients chronically infected with hepatitis C virus

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.

Interferon-induced thyroid dysfunction in chronic hepatitis C.

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.

Outbreak of Invasive Methicillin-Resistant Staphylococcus aureus Infection Associated With Acupuncture and Joint Injection

OBJECTIVE To describe an outbreak of invasive methicillin-resistant Staphylococcus aureus (MRSA) infection after percutaneous needle procedures (acupuncture and joint injection) performed by a single medical practitioner. SETTING A medical practitioners office and 4 hospitals in Perth, Western Australia. PATIENTS Eight individuals who developed invasive MRSA infection after acupuncture or joint injection performed by the medical practitioner. METHODS We performed a prospective and retrospective outbreak investigation, including MRSA colonization surveillance, environmental sampling for MRSA, and detailed molecular typing of MRSA isolates. We performed an infection control audit of the medical practitioners premises and practices and administered MRSA decolonization therapy to the medical practitioner. RESULTS Eight cases of invasive MRSA infection were identified. Seven cases occurred as a cluster in May 2004; another case (identified retrospectively) occurred approximately 15 months earlier in February 2003. The primary sites of infection were the neck, shoulder, lower back, and hip: 5 patients had septic arthritis and bursitis, and 3 had pyomyositis; 3 patients had bacteremia, including 1 patient with possible endocarditis. The medical practitioner was found to be colonized with the same MRSA clone [ST22-MRSA-IV (EMRSA-15)] at 2 time points: shortly after the first case of infection in March 2003 and again in May 2004. After the medical practitioners premises and practices were audited and he himself received MRSA decolonization therapy, no further cases were identified. CONCLUSIONS This outbreak most likely resulted from a breakdown in sterile technique during percutaneous needle procedures, resulting in the transmission of MRSA from the medical practitioner to the patients. This report demonstrates the importance of surveillance and molecular typing in the identification and control of outbreaks of MRSA infection.

Bartonella henselae Associated with Parinaud's Oculoglandular Syndrome

Bartonella henselae was recovered from the conjunctival scraping of a 38-year-old woman who presented with a 2-week history of tender preauricular lymphadenopathy and a 1-day history of a red left eye. Dry adherent colonies were observed on agar plates at 21 days of incubation, and the isolate was identified through conventional and molecular tests. Polymerase chain reaction (PCR) amplification of a specific region of the 16S rRNA gene and confirmation by a separate PCR reaction with hybridization of the product with a B. henselae-specific probe confirmed the isolate as B. henselae. This is the first reported isolation of the causative agent of cat scratch disease from ocular tissue in a patient with Parinauds oculoglandular syndrome.