Makoto Utsumi

Selection of human immunodeficiency virus type 1 variants with an insertion mutation in the p6gag and p6pol genes under highly active antiretroviral therapy

We detected several types of human immunodeficiency virus type 1 (HIV‐1) variants with an insertion mutation in the p6gag and p6pol genes in eight of twenty‐two (36.4%) patients who possessed drug‐resistant viruses under highly active antiretroviral therapy (HAART). It was characteristic that a conserved proline‐rich motif “PTAPP” in the N‐terminus of p6gag protein was completely or partially duplicated in all cases. Five among the eight cases were retrospectively investigated in terms of the occurrence of dynamic change in the gag gene between the inserted and wild‐type HIV‐1 in the course of HAART. The longitudinal analysis revealed the following: 1) The inserted‐type viruses were selected over the wild‐type during HAART in three cases in which the both types coexisted in the beginning of the therapy. 2) In two cases in which the inserted‐type HIV‐1 alone was detected before the beginning of HAART, the inserted‐type HIV‐1 alone was continuously detected during the therapy. The inserted‐type HIV‐1 was also detected in four of thirty‐nine (10.3%) therapy‐naive patients. However, the frequency of inserted‐type HIV‐1 detection in the HAART‐receiving patients is significantly higher than that in the therapy‐naive patients (P=0.02). These results suggest that this type of insertion mutation is a polymorphism of the p6gag and p6pol genes, however, it consequently gave an advantage on proliferation and/or survival of the HIV‐1 variant under the presence of antiretroviral drugs.

A novel method for detecting HIV-1 by non-radioactive in situ hybridization: application of a peptide nucleic acid probe and catalysed signal amplification.

A novel in situ hybridization (ISH) method for detecting human immunodeficiency virus‐1 (HIV‐1) was developed by applying a peptide nucleic acid (PNA) probe and a catalysed signal amplification (CSA) method. The PNA probe used in the present study possessed 15 base sequences of the HIV‐1 protease gene, and the 5′ end of the probe was labelled with the fluorescein isothiocyanate (FITC) molecule. The hybridized probe was detected by sequential reactions of the following antibodies and reagents: horseradish peroxidase (HRP)‐conjugated anti‐FITC antibody, biotinylated tyramide (first amplification), HRP‐labelled streptavidin, biotinylated tyramide (second amplification), and streptavidin‐conjugated Alexa 488. The signal of Alexa 488 was finally detected by fluorescence microscopy. HIV‐1‐related dotted signals were clearly obtained in HIV‐1 persistently infected cell lines, MOLT4‐IIIB and ACH‐2, and CD4‐positive T lymphocytes from AIDS patients. For light microscopy, HRP‐labelled streptavidin was reacted instead of streptavidin‐conjugated Alexa 488 at the final treatment, followed by diaminobenzidine as chromogen. This method can detect HIV‐1 in either blood smear samples or paraffin‐embedded autopsy tissue and is useful as a sensitive non‐radioactive method for in situ hybridization. Copyright

Prevalence of Drug‐Resistant Human Immunodeficiency Virus Type 1 in Therapy‐Naive Patients and Usefulness of Genotype Testing

In the present study, we performed genotypic drug‐resistance testing in 116 therapy‐naive human immunodeficiency virus type 1 (HIV‐1)‐infected patients between 1999 and 2002 at Nagoya National Hospital, Japan. The prevalence of drug‐resistant HIV‐1 with one or more major mutations significantly increased from 5.3% (4/75) in 1999–2001 to 17.1% (7/41) in 2002 (P=0.05), suggesting the spread of drug‐resistant HIV‐1. We identified a patient who possessed a protease (PR) inhibitor‐resistant HIV‐1 with a major mutation consisting of L90M before the initiation of therapy. The patient was administered zidovudine, lamivudine, and efavirenz as highly active antiretroviral therapy (HAART), as PR inhibitors were excluded based on the result of the drug‐resistance testing. The treatment succeeded in strongly suppressing the proliferation of drug‐resistant HIV‐1 and concomitantly increased CD4 cell counts. Thus, we conclude that drug‐resistance testing prior to the initiation of therapy is important for therapy‐naive patients to devise the optimum therapy regimen for each individual.

Failure to Detect Epstein-Barr Virus (EBV) DNA in Plasma by Real-Time PCR in a Case of EBV-Associated Posttransplantation Lymphoproliferative Disorder Confined to the Central Nervous System

We report here a patient who developed multiple central nervous system (CNS) space-occupying lesions 6 months after bone marrow transplantation from an HLA-matched unrelated donor. He had extensive chronic graft-versus-host disease and severe thrombocytopenia. Posttransplantation lymphoproliferative disorder (PTLD) was diagnosed after biopsy of the lesion was facilitated by the transfusion of 40 units of platelets. Epstein-Barr virus (EBV) DNA was not initially detected in the peripheral blood by real-time polymerase chain reaction, and the blood became positive for EBV at a low level only after more than 6 weeks had passed since the initial identification of detectable intracranial lesions. The patient died of cerebral herniation while donor leukocyte infusion was being prepared, and an autopsy confirmed the diagnosis of EBV-associated PTLD restricted to the CNS.

Prevalence of infection and genotypes of GBV-C/HGV among homosexual men

Since the discovery of GB virus‐C (GBV‐C) and hepatitis G virus (HGV), many studies have been performed. These viruses are now known to be parenterally, as well as sexually transmitted. A phylogenetic analysis also revealed that GBV‐C has five major genotypes: type 1 predominates in West Africa, type 2 in Europe and the United States, type 3 in parts of Asia, type 4 in Southeast Asia, and type 5 in South Africa. Despite the number of reports so far, there have been few large‐scale surveys of homosexual men to determine the prevalence of the GBV‐C/HGV infections. We examined the levels of GBV‐C/HGV viremia in 297 homosexual men who attended the Nagoya Lesbian and Gay Revolution held in Nagoya, Japan. Reverse transcription‐polymerase chain reaction (RT‐PCR)/nested PCR of the GBV‐C/HGV 5′‐non‐coding region (NCR), and base sequence analyses showed that the infection rate was 12.5%, and genotypes in this population were classified into type 2 (32%) and type 3 (68%). None were classified as types 1, 4, or 5 in this study. Our results indicate that the GBV‐C/HGV type 2 seen mainly in Europe and the US is spreading widely in Japan, especially in the Nagoya district.

The dual expression of minor and major bcr/abl chimeric mRNA in blast crisis of chronic myelogenous leukemia.

The hypothesis that minor bcr/abl fusion mRNA is produced in blast crisis of chronic myelogenous leukemia (CML) is examined. The RNA transcripts encoding the minor and major bcr/abl fused protein were detected by polymerase chain reaction (PCR) using RNA from peripheral blood or bone marrow cells of eight patients with blast crisis or accelerated phase of CML. The mRNA encoding for major bcr/abl was detected in all eight cases. In four patients, however, transcripts encoding for minor bcr/abl mRNA were detected, as well as major bcr/abl mRNA. The presence of minor bcr/abl mRNA was verified with the hybridization with a junction-specific probe and DNA sequencing analysis of PCR products. The appearance of minor bcr/abl fusion mRNA was associated with the lymphoblastic immunophenotype of the blast cells. In two of these four patients, samples of initial diagnosis of chronic phase of CML were available, which did not show minor bcr/abl transcript. We conclude that the appearance of minor bcr/abl mRNA transcript is associated with the terminal evolution of CML in lymphoblastic crisis.

An 11-Year Surveillance of HIV Type 1 Subtypes in Nagoya, Japan.

Abstract To monitor active HIV-1 transmission in Nagoya, Japan, we have been determining the subtypes of HIV-1 infecting therapy-naive individuals who have newly visited the Nagoya Medical Center since 1997. The subtypes were determined by phylogenetic analyses using the base sequences in three regions of the HIV-1 genes including gag p17, pol protease (PR) and reverse transcriptase (RT), and env C2V3. Almost all HIV-1 subtypes from 1997 to 2007 and 93% of all HIV-1 isolates in 2007 were subtype B. HIV-1 subtypes A, C, D, and F have been detected sporadically since 1997, almost all in Africans and South Americans. The first detected circulating recombinant form (CRF ) was CRF01_AE (11-year average annual detection rate, 7.7%). Only two cases of CRF02_AG were detected in 2006. A unique recombinant form (URF ) was first detected in 1998 and the total number of URFs reached 25 by year 2007 (average annual detection rate, 4.7%). Eleven of these 25 were detected from 2000 to 2005 and had subtypes AE/B/AE as determined by base sequencing of the gag p17, pol PR and RT, and env C2V3 genes (average annual detection rate, 3.7%). Unique subtype B has been detected in six cases since 2006. All 17 of these patients were Japanese. Other recombinant HIV-1s have been detected intermittently in eight cases since 1998. During the 11-year surveillance, most HIV-1s in Nagoya, Japan were of subtype B. We expect that subtype B HIV-1 will continue to predominate for the next several years. Active recombination between subtype B and CRF01_AE HIV-1 and its transmission were also shown.

Delayed HIV-1 infection of CD4+ T lymphocytes from therapy-naïve patients demonstrated by quantification of HIV-1 DNA copy numbers.

Measuring the amount of HIV‐1 DNA in infected cells is important to estimate the size of the viral reservoir in patients. However, the clinical impact of the intracellular viral DNA level remains unclear. The present study examines the clinical significance of the HIV‐1 DNA level in peripheral CD4+ T lymphocytes from 21 therapy‐naïve patients. HIV‐1 DNA levels in purified peripheral CD4+ T lymphocytes were measured by the real‐time PCR method using the Roche LightCycler system that can detect 200 copies/106 cells. We detected intracellular HIV‐1 DNA in 15 (71.4%) of 21 patients at levels ranging from 270 to 98,120 copies/106 CD4+ cells, with a median of 2,220 copies/106 cells. We also found HIV‐1 DNA that was below the detection limit in the remaining 6 patients, although 8,800–150,000 copies/ml of HIV‐1 RNA were detected in plasma. Circular HIV‐1 DNA was not detected in 5 of 6 cases, suggesting that reverse transcription in CD4+ T lymphocytes of these cases was not active. Thus, delayed HIV‐1 infection of CD4+ T lymphocytes was demonstrated in these patients. The level of HIV‐1 DNA in peripheral CD4+ T lymphocytes indicates the clinical status of therapy‐naïve patients.

Achievement of a complete cytogenetic response with hydroxyurea in a patient with chronic myelogenous leukemia

Hydroxyurea rarely produces a complete cytogenetic remission in patients with chronic myelogenous leukemia (CML). In this report, we describe one case of the CML patient who achieved complete cytogenetic remission (no Ph chromosome in 20-25 metaphase cells) by treatment with hydroxyurea alone. By the fluorescent in situ hybridization (FISH) methodology using bcr/abl specific translocation probe, sequential bone marrow specimens from the patient showed the characteristic 9;22 translocation at a higher rate (9-10%) than the normal control range (2.49-4.88%) at the time of complete cytogenetic remission. Thus, it is suggested that FISH is a more sensitive method to detect the bcr/abl fusion gene than conventional cytogenetic analysis for the detection of minimal residual disease in CML.

Measurement of Miconazole Serum Concentration in High-performance Liquid Chromatography.

Miconazole (MCZ) serum concentration was measured by high-performance liquid chromatography (HPLC). We assessed whether the internal standard method produced on intra-assay error, and found that the method gave more precise and more reproducible results than absolute calibration curve method. With 0.5μg/ml of MCZ, the coefficient of variation produced by that method was 3.41%, whereas that of the absolute calibration curve method was 5.20%. The concentrations of absolute calibration curve method showed higher values than the internal standard method. This indicated that the internal standard method was far more precise in measuring the MCZ serum concentrations than was the absolute calibration curve method.