Evaluation of Geenius HIV-1/2 Confirmatory Assay for the confirmatory and differential diagnosis of HIV-1/HIV-2 in Japan and reliability of the Geenius Reader in the diagnosis of HIV-2.

BACKGROUND: NEW LAV BLOT I and II (LAV I and LAV II), they were only option for human immunodeficiency virus (HIV) confirmatory test, following HIV screening test using HIV Ag/Ab combination test in Japan. We evaluated the performance of Geenius HIV-1/2 Confirmatory Assay (Geenius), both as a confirmatory test and for differentiation between HIV-1 and HIV-2, in comparison with LAV I and LAV II. METHODS: Eighty-nine HIV-1-positive plasma specimens, one anti-HIV-1 low-titer performance panel, 10 seroconversion panels, and two anti-HIV-1/2 combo performance panels were tested. The results were read with the Geenius Reader and by visual reading. RESULTS: All 89 HIV-1-positive plasma specimens were identified as HIV-1-positive using Geenius; this 100% success rate was superior to that with LAV I (95.5% using WHO criteria, 98.9% using CDC criteria). The HIV-1-positive specimens showed low cross-reactivity with HIV-2 lines in Geenius. The sensitivity of Geenius for HIV-1 detection was the same as or greater than that of LAV I, but less than that of Genscreen HIV Ag-Ab ULT, in our analysis of the commercial performance and seroconversion panels. In contrast, five of the 13 HIV-2-positive specimens that had been identified as HIV-positive untypable by visual reading because of their cross-reactivity to HIV-1 lines were successfully identified by the Geenius Reader as HIV-2-positive with cross-reactivity. CONCLUSIONS: Geenius provides strong performance for HIV confirmatory tests and HIV-1 differentiation tests. However, when visual reading is used, its performance in HIV-2 differentiation is less reliable. Because HIV-2 infection has been sporadically reported in Japan, the use of the Geenius Reader is preferable to ensure more reliable HIV-1/HIV-2 differentiation.

The spontaneous clearance of hepatitis E virus (HEV) and emergence of HEV antibodies in a transfusion-transmitted chronic hepatitis E case after completion of chemotherapy for acute myeloid leukemia.

A 64-year-old woman was infected with hepatitis E virus (HEV) during chemotherapy for leukemia. By retrospective analyses of stored serum from the blood products and the patient, the source of the infection was determined to be platelet concentration (PC) transfused during chemotherapy. The partial nucleotide sequence of the HEV strain isolated from the donated PC and that from the patient's sera was identical and was subgenotype 3b. Clinical indicators such as alanine aminotransferase, HEV RNA titer, and anti-HEV antibodies in the serum were investigated from the beginning of the infection until 1 year after the termination of HEV infection. HEV RNA had propagated over 6 months and then cleared spontaneously after the completion of chemotherapy. Anti-HEV antibodies appeared in the serum just before the clearance of HEV RNA. Interestingly, HEV RNA was detected in the patient's urine, spinal fluid, and saliva. The HEV RNA titers in those samples were much lower than in the serum and feces. No renal, neurological, or salivary gland disorders appeared during the follow-up. We observed virological and biochemical progress and cure of transfusion-transmitted chronic hepatitis E in the patient despite an immunosuppressive status during and after chemotherapy against hematological malignancy.

Optimal titer of anti-HBs in blood components derived from donors with anti-HBc.

BACKGROUND: The ultimate strategy to cope with transfusion-transmitted hepatitis B virus (HBV) infection caused by transfusion of blood from donors with historical HBV infection is to reject all donors having anti-HBV core antigen (anti-HBc). However, this strategy would result in a huge loss of blood donors and subsequent blood inventory collapse. On the other hand, anti-HBc-positive blood is reportedly not infectious when containing more than 100 mIU/mL of anti-HBV surface antigen (anti-HBs). STUDY DESIGN AND METHODS: In Japan, anti-HBc-positive blood has been used for transfusion if it contained 200 mIU/mL or more of anti-HBs. First, to verify the screening policy, clinical outcomes for transfusion of such blood were analyzed for the 2008-2012 period. Second, human hepatocyte-repopulated severe combined immunodeficiency mice were inoculated with HBV preincubated with varying doses of anti-HBs, then viremic status was followed. The effects of anti-HBs across different HBV genotypes were also investigated. RESULTS: Twenty-three transfusion-transmitted HBV infections related to anti-HBc-positive blood components were identified. None of the blood responsible for these cases contained 200 mIU/mL or more of anti-HBs. When 100 µL of plasma containing 104 copies of HBV and 20 mIU of anti-HBs was injected into severe combined immunodeficiency mice, no viremia was detected within 13 weeks. Genotype C anti-HBs was capable of total inhibition of genotype A HBV replication, whereas genotype A anti-HBs inhibited genotype C HBV to a lesser extent. CONCLUSION: Anti-HBc-positive blood containing 200 mIU/mL or more of anti-HBs appears safe as a transfusion component. HBV vaccination seems effective between HBV genotypes A and C.

Evaluation of in vitro screening and diagnostic kits for hepatitis B virus infection.

BACKGROUND: For the diagnosis of hepatitis B virus (HBV) infection, the detection and quantification of hepatitis B surface antigen (HBsAg) and HBV DNA are used. Several kits are available for this purpose, and there is a growing need for the evaluation of these kits because their performance may be affected by HBV genotype- or strain-specific polymorphisms. OBJECTIVES AND STUDY DESIGN: In this study, we used International Standards and the established regional reference panel to evaluate the performance of two HBV DNA quantitative kits, five HBsAg qualitative kits, seven HBsAg quantitative kits and three rapid immune-chromatographic tests for HBsAg. RESULTS: The quantification values of two HBV DNA quantitative kits exhibited excellent correlation. In the evaluation of HBsAg qualitative and quantitative kits, the titers of several specimens in the HBV-positive panel were below the detection limits of a few kits, and the specimens were determined as HBV-negative. Notably, the quantitative kit results exhibited low correlation values. However, when these data were analyzed for each genotype, the correlations improved. These results suggest that the HBsAg quantification data are influenced by HBV genotypes. The novel rapid immune-chromatographic test exhibited the comparable level of sensitivity to the HBsAg quantitative kits. CONCLUSIONS: We evaluated the performance of kits for the detection of HBV infection. The HBV DNA quantification data correlated with an excellent agreement, whereas the HBsAg quantification data were affected by HBV genotype. Such evaluations will be useful for estimating the quality of currently available and new HBV assay kits, and for the quality control of these kits.

Evaluation of in vitro screening and diagnostic kits for hepatitis C virus infection.

BACKGROUND: To detect infection by hepatitis C virus (HCV), a reliable kit with high sensitivity and specificity is indispensable. Detection kits for anti-HCV antibodies (anti-HCV) are used for screening, and quantification kits for HCV RNA and core antigen are used for definite diagnosis of HCV infection. OBJECTIVES: We evaluated the performance of these kits using International Standards and a regional reference panel with HCV negative and positive specimens. STUDY DESIGN: In vitro diagnostic kits (10 anti-HCV, two HCV RNA, and three HCV core antigen) were included. RESULTS: Nearly all specimens in the regional reference panel were correctly identified by all anti-HCV detection kits (one false-positive was observed in one kit). Both HCV RNA quantification kits also correctly identified and quantified HCV RNA titers, without genotype-specific differences. Among the HCV core antigen kits, International Standard values were inconsistent. The sensitivities of these kits were insufficient to detect HCV in positive specimens in the regional reference panel. CONCLUSIONS: In vitro diagnostic kits assessing anti-HCV and HCV RNA have sufficient sensitivities and specificities to screen and detect HCV infection. However, HCV core antigen quantification kits have some limitations in their sensitivities and consistencies for diagnosis of HCV infection. Quality control with International Standards and a regional reference panel is important to maintain the performances of diagnostic kits for HCV infection and to verify the clinical reliability of these kits.

Persistent symptomatic parvovirus B19 infection with severe thrombocytopenia transmitted by red blood cell transfusion containing low parvovirus B19 DNA levels.

BACKGROUND: Transfusion-mediated human parvovirus B19 (PVB19) infection is rare but often causes severe hematologic disorders. In Japan, routine blood donor screening for PVB19 antigen (detection sensitivity, 106.4 IU/mL) using a chemiluminescent enzyme immunoassay (CLEIA) was introduced in 2008. However, there is no consensus on the minimal infectious dose of PVB19 permissible for red blood cells (RBCs). CASE REPORT: A 64-year-old man, who had received hemodialysis for diabetic nephropathy for 5 years, underwent an RBC transfusion for anemia caused by hemorrhagic enterocolitis. He developed persistent high fever and progressive thrombocytopenia. He was diagnosed with PVB19 infection when a marrow examination showed giant erythroblasts, and his serum was positive for PVB19 DNA. His serum was negative for PVB19 immunoglobulin (Ig)M and IgG before transfusion, but positive for both after transfusion. This PVB19 infection was deemed to be transmitted by the RBC transfusion because low levels of PVB19 DNA (1.10 × 104 IU/mL) were detected in one of the blood donors. A DNA homology test of PVB19 showed complete genomic identity between the virus in the donor and our patient. CONCLUSION: We report a patient who developed persistent PVB19 infection from an RBC transfusion containing low levels of PVB19. This is the second case of transfusion-mediated PVB19 infection since the introduction of CLEIA in 2008. Transmission may occur in immunocompromised patients lacking PVB19-neutralizing antibodies. The report of further such cases will allow the establishment of minimal threshold values and more effective screening tests for PBV19 transmission through RBC products.

Unique clinical courses of transfusion-transmitted hepatitis E in patients with immunosuppression.

BACKGROUND: The high prevalence of specific immunoglobulin G for hepatitis E virus (HEV) in Japanese people raises the possibility of a high incidence of HEV-viremic blood donors and therefore frequent transfusion-transmitted HEV (TT-HEV). STUDY DESIGN AND METHODS: TT-HEV cases established in Japan through hemovigilance and those published in the literature were collected. Infectivity of HEV-contaminated blood components and disease severity in relation to immunosuppression were investigated. RESULTS: Twenty established TT-HEV cases were recorded over the past 17 years. A lookback study verified that five of 10 patients transfused with known HEV-contaminated blood components acquired HEV infection. The minimal infectious dose of HEV through transfusion was 3.6 × 104 IU. Nine of the 19 TT-HEV cases analyzed had hematologic diseases. Only two cases showed the maximal alanine aminotransferase level of more than 1000 U/L. Two patients with hematologic malignancy and two liver transplant recipients had chronic liver injury of moderate severity. CONCLUSION: The infectivity of HEV-contaminated components was 50%. Immunosuppression likely causes the moderate illness of TT-HEV, but it may lead to the establishment of chronic sequelae. Transfusion recipients, a population that is variably immunosuppressed, are more vulnerable to chronic liver injury as a result of TT-HEV than the general population is as a result of food-borne infection.

Sequence analysis of two variable cytomegalovirus genes for distinction between transfusion- and breast milk-transmitted infections in a very-low-birthweight infant.

BACKGROUND: Cytomegalovirus (CMV) infections in very-low-birthweight infants can lead to serious clinical consequences. When CMV-related symptoms occur after transfusion, CMV transmission is often attributed to the transfusion products rather than to breast milk. However, it is sometimes difficult to distinguish between transfusion-transmitted and breast milk-transmitted CMV infections. PATIENT AND METHODS: A patient was born at 27 gestational weeks with a weight of 689 g. He was transfused with leukoreduced red blood cells (LR-RBCs), which were later found to be CMV seropositive and CMV DNA positive. He was also fed with CMV DNA-positive breast milk. Thereafter, he developed CMV disease with thrombocytopenia and jaundice. To determine the route of transmission, we analyzed the sequences of two variable CMV genes, UL139 and UL146, by direct sequence analysis. We also performed deep sequence analysis to determine whether there were polyclonal CMV strains in the LR-RBCs transfused. RESULTS: CMV DNA sequence-matching rates for the LR-RBCs and the patient's blood were 64.6% for the UL139 gene and 68.6% for the UL146 gene. In contrast, the sequences of these genes in the patient's blood were 100% matched with those in the breast milk. Furthermore, by deep sequence analysis, the CMV strain found in the patient's blood was not detected in the LR-RBCs transfused. CONCLUSION: The results indicate that the pathogenic CMV strain was transmitted through breast milk, which is consistent with the claims that transfusion-transmitted CMV infection due to leukoreduced blood products is uncommon.

Cytomegalovirus (CMV) seroprevalence in Japanese blood donors and high detection frequency of CMV DNA in elderly donors.

BACKGROUND: The current prevalence of cytomegalovirus (CMV) in Japan and the risk of CMV transfusion transmission are unknown in the era of seronegative leukoreduced blood components. STUDY DESIGN AND METHODS: We measured CMV-specific immunoglobulin (Ig)M and IgG in 2400 samples of whole blood collected from 12 groups of blood donors categorized by sex and age at 10-year intervals from their teens to their 60s. We also tested for CMV DNA using polymerase chain reaction in the cellular fractions of all samples. RESULTS: We found that 76.6% of blood donors were CMV seropositive. The seroprevalences among donors in their 20s and 30s were 58.3 and 73.3%, respectively. We detected CMV DNA in the cellular fraction of 4.3% of samples from donors in their 60s and in 1.0% of samples from donors younger than 60 years. None of the 562 seronegative samples was DNA positive. Furthermore, 14% of DNA-positive samples also contained DNA in the plasma fraction, and two of five such samples were derived from donors in their 60s. Leukoreduced plasma components derived from donations with CMV DNA in plasma samples also contained a relevant amount of CMV DNA. CONCLUSION: The seroprevalence of CMV among Japanese blood donors of child-bearing age has not changed over the past 15 years. Latent CMV becomes reactivated more frequently among elderly donors than among younger donors. A proportion of them have free CMV DNA in their plasma fraction, which could not be diminished by leukoreduction. The risk of transfusion-transmitted CMV infection in blood with plasma CMV DNA should be determined.

Prevalence of amino acid mutation in hepatitis C virus core region among Japanese volunteer blood donors.

It is not known whether there is a trend of increasing or decreasing incidence of new hepatitis C virus (HCV) infections in Japan. From the treatment point of view, it is important to verify HCV genotypes and the prevalence of treatment-resistant clones of HCV. At the Japanese Red Cross blood centers, all blood samples obtained from blood donation have been screened using serological methods and the minipool nucleic acid amplification testing. One hundred and fourteen donors have been identified over the past 10 years to be HCV RNA-only positive without detectable anti-HCV and were considered to be in the acute phase of HCV infection. There was a trend of decreasing incidence of such new infections among the blood donors. HCV RNA-only-positive samples were examined further for genotyping and HCV RNA quantitation. Genotype 2 (2a plus 2b) was predominant (78.2%) among them followed by genotype 1b (21.2%). Direct sequencing was carried out to detect the possible treatment-resistant mutant clones 70Q and 91M, clones with amino acid substitutions at positions 70 and 91 of the HCV core protein, respectively. 70Q and 91M were found regularly in donors with genotype 1b, but not in those with other genotypes. No particular endemic areas for the mutant clones were identified. There was no significant difference in the mean viral titer between the 70Q mutant type and the non-70Q wild-type. Even in newly infected people, the mutant clone 70Q was detected frequently.

Novel DNA sequence isolated from blood donors with high transaminase levels.

AIM: In Japan, the etiology of 10-20% of cases of acute hepatitis remains unclarified. This study was conducted to verify the agent causing non-A-E hepatitis. METHODS: Serum samples from 500 blood donors with elevated alanine aminotransferase (ALT) levels were screened by polymerase chain reaction using primers constructed from conserved areas of RNA virus helicase. The sequence obtained was investigated for viral properties. RESULTS: Four blood samples were found to contain a novel DNA sequence of 9496 bp, which was designated KIs-V. KIs-V was sensitive to the restriction enzyme SalI and BstXI. Rolling-circle amplification produced an excessive amount of KIs-V DNA. In sucrose density gradient ultracentrifugation, KIs-V banded at a 1.158-g/cm(3) density. Detergent treatment increased the density of KIs-V. There was no KIs-V DNA amplification from human leukocyte DNA. Serial filtration suggested that KIs-V was included in a 30-50-nm size particle. In silico analysis revealed that KIs-V contained 13 potential genes, none of which showed homology to any viral proteins reported. One gene showed similarity to a DNA polymerase domain. Strong signals for transcription initiation and a CpG island were identified. The nucleotide composition of KIs-V showed a characteristic feature of circular DNA genomes that contain a replication origin and a terminus. In a preliminary study, KIs-V was frequently identified among hepatitis E virus antibody positive individuals with elevated ALT levels. CONCLUSION: A new sequence KIs-V was isolated from blood donors with elevated ALT levels. It was suggested that KIs-V is a double-stranded circular DNA genome derived from a novel category of enveloped viruses.

Symptomatic parvovirus B19 infection caused by blood component transfusion.

BACKGROUND: Although a risk of transfusion-transmitted human parvovirus B19V (TT-B19V) infection has been a concern, there have been very few reports of clinically relevant TT-B19V caused by the transfusion of a B19V-containing blood component. It has therefore been a matter of debate whether a universal B19V screening with an appropriate sensitivity is required. STUDY DESIGN AND METHODS: Through the Japanese Red Cross hemovigilance system, clinical reports on possible TT-B19V were collected from 1999 to 2008, during which B19V donor screening (sensitivity, 10(10) IU/mL) was conducted and repository blood samples from donors were available. RESULTS: Eight patients with TT-B19V caused by component transfusion have been identified. Four patients developed sustained anemia and pure red blood cell (RBC) aplasia and one patient developed pancytopenia. The underlying diseases in these five patients were either hematologic malignancy or hemolytic diseases. The viral loads of the responsible components for these cases ranged from 10(3) to 10(8) IU/mL. Two patients who underwent surgical treatment without any hematologic disorder exhibited only moderate symptoms. The B19V DNA sequence identity between a patient and the linked blood donor was confirmed in five of the eight patients. All of the components responsible for the eight cases were positive for anti-B19V immunoglobulin (Ig)M. CONCLUSION: Vulnerability to serious B19V-related hematologic disorders depended on the patient's underlying disease state of an enhanced erythropoiesis, not on the viral load of the component transfused. To prevent clinically relevant TT-B19V, a strategy is suggested in which patients at risk of acquiring RBC aplasia or pancytopenia are targeted.

Influence of preparation method on polynucleotide conformation and pharmacological activity of lipoplex.

Conformations of polyinosinic acid [poly(I)] and polycytidylic acid [poly(C)] in liposomes (lipoplex) were investigated by both circular dichroism (CD) spectroscopy and fluorescence resonance energy transfer (FRET) measurements, and compared with those in aqueous solution. The results indicate that poly(I) and poly(C) take double-stranded structure in aqueous solution at pH 6.5-7.5 in the presence of NaCl at higher concentration than 50mM. Although lipoplex was prepared without NaCl to avoid aggregation of lipoplex particles, poly(I) and poly(C) were double-stranded in pre-mixed poly(I)/poly(C) lipoplex (pre-mixed LIC), prepared by adding a mixed solution of poly(I) and poly(C) to the cationic liposomes. However, poly(I) and poly(C) did not take double-stranded structure in separately mixed LIC, prepared by separate addition of poly(I) solution and poly(C) solution to the cationic liposomes. The physicochemical properties (particle diameter and zeta potential) of pre-mixed LIC and separately mixed LIC were not different, but the anti-proliferative effect of pre-mixed LIC on human epidermoid carcinoma A431 cells was about eight times greater than that of separately mixed LIC. Our results indicate that polynucleotide conformation in lipoplex is markedly influenced by the preparation method, and the polynucleotide conformation in lipoplex has a substantial effect on pharmacological activity.