Prolonged use of tenofovir in HIV/hepatitis B virus (HBV)-coinfected individuals does not lead to HBV polymerase mutations and is associated with persistence of lamivudine HBV polymerase mutations.

OBJECTIVES: The aim of the study was to identify and characterize hepatitis B virus (HBV) polymerase gene mutations associated with ongoing HBV replication in HIV/HBV-coinfected individuals receiving tenofovir (TDF). METHODS: This retrospective cross-sectional study identified 28 HIV/HBV-coinfected individuals who had received TDF for at least 3 months. All patients had samples available while receiving TDF (on-TDF), and 24 also had samples available prior to treatment (pre-TDF). Case records were reviewed to obtain clinical and virological data at the times of sampling (+/-3 months). The HBV DNA of all samples was amplified using polymerase chain reaction (PCR), and the polymerase region of PCR-positive samples was sequenced and compared with reference HBV data. RESULTS: Of the pre-TDF samples, 15 of 24 (63%) were HBV PCR positive. Of the on-TDF samples, four of 28 (14%) were HBV PCR positive (mean time on TDF 13.5 months; range 3-23 months). Lamivudine (3TC)-resistance mutations were detected in three of four (75%) of these viraemic samples. The previously identified putative TDF-resistance mutations, rtA194T+rtL180M+rtM204V, were not detected in any individual. CONCLUSIONS: Unique mutations in the HBV polymerase gene associated with TDF resistance are rare in HIV/HBV coinfection. 3TC-resistance mutations persist and a significant proportion of patients are HBV PCR positive despite the addition of TDF.

Ten-year review of disease pattern from percutaneous renal biopsy: an experience from a paediatric tertiary renal centre in Hong Kong.

OBJECTIVE: To study the childhood renal disease pattern based on the renal biopsy histology in a local paediatric tertiary renal centre. DESIGN: Retrospective study. SETTING: Department of Paediatrics and Adolescent Medicine, Princess Margaret Hospital, Hong Kong. PATIENTS: All patients who underwent real-time ultrasound-guided closed renal biopsy from 1 April 1997 to 31 March 2007 were included. RESULTS: A total of 209 renal biopsies were performed, 162 on native kidneys and 47 on grafts. In the native group, major indications were renal manifestations secondary to systemic diseases (34%), followed by idiopathic nephrotic syndrome (28%) and haematuria (27%). In 94% the histopathology revealed glomerular diseases. Among the primary glomerular diseases, thin glomerular basement membrane disease, immunoglobulin A nephropathy, minimal change disease, and focal segmental glomerulosclerosis accounted for most. In all, 37% of patients with steroid-resistant nephrotic syndrome had focal segmental glomerulosclerosis and its relative incidence was increased when compared to previous studies. Minimal change disease and minimal change disease with mesangial immunoglobulin M deposits accounted for the majority of steroid dependent and frequent relapsers. Among patients with isolated microscopic haematuria, 73% had thin glomerular basement membrane disease, while patients with concomitant haematuria and proteinuria had a wide variety of pathology. In the kidney graft group, acute graft dysfunction was due to acute rejection in 38% of the patients, followed by calcineurin inhibitor toxicity in 14%. Chronic allograft nephropathy caused chronic allograft dysfunction in the majority of cases. Post-transplant proteinuria was caused by recurrence of the primary renal disease in all of our patients. CONCLUSION: This study provides updated epidemiological information for childhood renal disease and a change in the pattern of disease was observed.

Multiple outbreaks of Norwalk-like virus gastro-enteritis associated with a Mediterranean-style restaurant.

The role of diverse infectious agents, particularly Norwalk-like viruses (NLV), in three successive gastro-enteritis outbreaks in one setting (a restaurant) was evaluated. Methods included standard bacteriological tests, specific tests for Escherichia coli, tests for verocytotoxins, electron microscopy (EM) for viruses and reverse transcription-PCR (RT-PCR) methodology for NLV. No pathogenic bacteria were detected. Verocytotoxin genes, although detected by PCR in the first outbreak, could not be confirmed in the E. coli isolated, so they did not appear to be of significance. NLV was the main agent detected in each of the three outbreaks. DNA sequencing and phylogenetic analysis of the amplified products obtained from the RT-PCR positive specimens indicated that only one NLV strain was involved in each outbreak, but the NLV strains responsible for the three outbreaks were different from each other. PCR technology for detection of NLV proved highly sensitive, but failed to detect one specimen which was positive by EM. The restaurant associated with the outbreaks is a Mediterranean-style restaurant where food from a common platter is typically eaten with fingers. The findings indicate that NLV was introduced by guests or staff and was not due to a long-term reservoir within the setting.

Jervell-Lange Nielsen syndrome in a Pakistani family.

Congenital long QT syndrome is a rare hereditary disease that is related to the dysfunction of ion channels in cardiac cells. We report on a very rare case of its autosomal recessive form--the Jervell-Lange Nielsen syndrome--in a Pakistani family, which was diagnosed after the incidental finding of bradycardia in a newborn baby girl. We discuss the range of presentations in neonates; the importance of strong suspicion of the syndrome and family screening; the use of the diagnostic criteria and genetic tests; and the different management strategies.

Identification and protein analysis of polyomavirus assembly intermediates from infected primary mouse embryo cells.

A method is described for the isolation of polyoma virus assembly intermediates from infected mouse embryo cells. Sucrose gradient profiles revealed the presence of 90 S, 200 S, and 240 S intermediates. These intermediates were shown to be sensitive to a number of factors: ionic condition of the isolation buffer, presence of chelating agents and nonionic detergents during isolation, and sonication of nuclei during extraction of intermediates. Pulse-chase experiments demonstrated that the order of formation of the intermediates to be 90 S----240 S, with the 200 S particles as a possible intermediate form linking the 90 S and 240 S particles. Viral structural proteins VP1, VP2, and VP3 were shown to be present on all three intermediates, but the ratio of each protein varied on each intermediate species. Two-dimensional gel electrophoresis demonstrated that the distribution of the VP1 isoelectric focusing species were different among the three intermediates. Histone H1 was found exclusively with the 90 S species.

Improved infectivity of reassembled polyoma virus.

Polyoma virus was dissociated into capsomeres (18, 12, and 5S) and a DNA-protein complex (48S) with the Ca2+ chelator, ethyleneglycol-bis-N,N'-tetraacetic acid, and the reducing agent, 2-mercaptoethanol. The reaction was maintained at pH 5.0. Reassembly of the dissociated components to complete virions was accomplished by dialyzing these components overnight at 4 degrees C against the reassembly buffer containing CaCl2, dimethylsulfoxide, Triton X-100, and 0.01 M Tris-acetic acid (pH 5.0). Reconstructed particles ranged from 240S complete virions to lighter intermediate species. Approximately 25% of the dissociated particles could be physically reassembled to complete virions. These virions regained 12.5% of their hemagglutination ability and as much as 6.7% of their original infectivity. The infectivity of these reassembled particles represented a 100-fold increase in infectivity compared with that of the particles that were dissociated and reassembled at pH 7.4. Biochemical analysis showed that the polyoma viral receptor of the virions reassembled at pH 7.4 was greatly reduced, whereas virions reassembled at pH 5.0 retained their receptor. Reassembly could be further improved by additions of either exogenous capsomeres or DNA-protein complex to the reassembly reaction mixture.

Generation of capsids from unstable polyoma virions.

Polyomavirus was purified from infected mouse cell lysates under mild physiological conditions. When analyzed in a sucrose gradient, a major virus peak (240S) was identified. This sucrose-isolated virus could be divided into two populations based on its stability to CsCl gradient centrifugation. Members of the unstable population were shown to eject their DNA cores when subjected to CsCl gradient centrifugation, forming empty capsids, whereas the stable population was unaffected by the same CsCl treatment. Formaldehyde fixation of the 240S virus particles stabilized the virions and prevented ejection of DNA and generation of empty capsids. When formaldehyde-fixed 240S virus was examined with the electron microscope, only full virions were observed. These results indicate that polyoma capsids are not preformed in vivo, but instead are generated when infected cell lysates are subjected to harsh CsCl purification procedures.

Bacteriological and molecular analysis of rifampin-resistant Mycobacterium tuberculosis strains isolated in Australia.

To develop a better understanding of the epidemiology and molecular biology of rifampin-resistant Mycobacterium tuberculosis strains in Australia, 50 clinical isolates (33 rifampin-resistant and 17 rifampin-sensitive strains) cultured between 1990 and 1997 were analyzed by a number of bacteriological and molecular techniques. Examination of the drug resistance profiles of the 33 rifampin-resistant isolates revealed that 91% were resistant to rifampin in combination with resistance to isoniazid, 88% were resistant to rifampin on first isolation, and 81% showed cross-resistance with rifabutin. On the basis of the demographic data provided for the patients infected with the rifampin-resistant strains, 90% of the patients were born overseas. Of these patients, 64% developed clinical symptoms within 5 years of residence in Australia. On a molecular level, analysis of the rpoB gene revealed that 97% of the rifampin-resistant isolates had missense mutations within a conserved region of the gene, and eight types of missense mutations were detected. Of the 31 rifampin-resistant isolates that were typed by restriction fragment length polymorphism (RFLP) analysis, 28 distinct patterns were obtained by RFLP analysis with IS6110, and three clusters of genetically related isolates were identified. All isolates within the clusters were from patients who were born overseas and who had the same country of origin. The results from this study provide an overview of the current situation of rifampin resistance in Australia and can serve as a basis for continued monitoring of drug-resistant M. tuberculosis strains isolated within the country.

Characterization of Mycobacterium tuberculosis strains from Vietnamese patients by Southern blot hybridization.

A total of 41 Mycobacterium tuberculosis strains from patients of Vietnamese origin were analyzed by Southern blot hybridization with two different probes, IS6110 (Otal, I., et al., J. Clin. Microbiol. 29:1252-1254, 1991; Ross, B. C., et al., J. Clin. Microbiol. 30:942-946, 1992; Thierry, D., et al., J. Clin. Microbiol. 28:2668-2673, 1990; van Soolingen, D., et al., J. Clin. Microbiol. 29:2578-2586, 1991) and pTBN12 (Ross, B. C., et al., J. Clin. Microbiol. 30:942-946, 1992). The restriction fragment patterns of nine of these strains were virtually identical when the pTBN12 probe was used; five strains had a single copy of IS6110, and four strains failed to hybridize with the IS6110 probe. This relatively high frequency of strains with no or one copy of IS6110 suggests that the usefulness of IS6110 for epidemiological study may be limited in certain populations.

Heminested multiplex reverse transcription-PCR for detection and differentiation of Norwalk-like virus genogroups 1 and 2 in fecal samples.

The present study describes a heminested multiplex reverse transcription (RT)-PCR assay which enables simultaneous detection and differentiation of Norwalk-like virus (NLV) genogroups from clinical fecal samples without the need to perform sequencing or hybridization. The assay developed was able to detect concentrations of fewer than 100 viral particles per 5 microl of clarified fecal extract and could differentiate the two genogroups with a specificity of 100%. Although the multiplex RT-PCR assay failed to detect NLV in about 3% of the fecal samples which were NLV positive by electron microscopy (EM), the assay was approximately six times more sensitive than EM for NLV detection.

Adolescent twin sisters with severe acute respiratory syndrome (SARS).

A novel coronavirus-associated communicable respiratory disease, severe acute respiratory syndrome (SARS), spread worldwide after an outbreak in Guangdong Province of the People's Republic of China in November 2002. Since late February 2003, there has been an epidemic in Hong Kong involving both adult and pediatric patients. The clinical course, intensive care, and outcome of adolescent twin sisters with SARS are described. Adolescents infected with SARS may develop severe illness as adults, and close monitoring for disease progression in terms of both clinical and radiologic deterioration is warranted.