JC polyomavirus nephropathy confirmed by using an in‐house polymerase chain reaction method

We report the case of an isolated JC virus (JCV) infection, without co‐infection by polyoma BK virus (BKV), associated with nephropathy 4 years after kidney transplantation. Clinical suspicion followed the observation of a decrease in estimated glomerular filtration rate (eGFR) and a renal allograft biopsy revealing polyomavirus‐associated tubulointerstitial nephritis and positivity for SV40. An in‐house real‐time polymerase chain reaction assay, targeting the presence of JCV and the absence of BKV in biopsy tissue, confirmed diagnosis. Thirteen months after diagnosis, and following therapeutic measures, eGFR remains stable.     

Detection of CAPN10 copy number variation in Thai patients with type 2 diabetes by denaturing high performance liquid chromatography and real‐time quantitative polymerase chain reaction

Aims/Introduction

A combination of multiple genetic and environmental factors contribute to the pathogenesis of type 2 diabetes. Copy number variations (CNVs) are associated with complex human diseases. However, CNVs can cause genotype deviation from the Hardy–Weinberg equilibrium (HWE). A genetic case–control association study in 216 Thai diabetic patients and 192 non-diabetic controls found that, after excluding genotyping errors, genotype distribution of calpain 10 (CAPN10) SNP44 (rs2975760) deviated from HWE. Here, we aimed to detect CNV within the CAPN10 SNP44 region.

Materials and Methods

CNV within the CAPN10 SNP44 region was detected using denaturing high-performance liquid chromatography, and the results confirmed by real-time quantitative polymerase chain reaction with SYBR Green I.

Results

Both methods successfully identified CNV in the CAPN10 SNP44 region, obtaining concordant results. Correction of genotype calling based on the status of identified CNVs showed that the CAPN10 SNP44 genotype is in good agreement with HWE (P > 0.05). However, no association between CNV genotypes and risk of type 2 diabetes was observed.

Conclusions

Identified CNVs for CAPN10 SNP44 genotypes lead to deviation from HWE. Furthermore, both denaturing high-performance liquid chromatography and real-time quantitative polymerase chain reaction are useful for detecting CNVs.     

Hepatitis B virus infection is associated with deletion of chromosome 8p in multiple myeloma

Serological analyses within epidemiological cohort and case‐control studies indicate to an association between HBV infection and risk of multiple myeloma (MM). To verify the relationship with an independent approach, we investigated the correlation between HBV positivity and chromosomal aberrations within 680 patients of the National Center for Tumor Diseases Heidelberg for which the serological HBV status (HBsAg and anti‐HBc) and FISH data for five gains (1q21, 9q34, 11q23, 15q22, 19q13), five losses (6q21, 8p21, 13q14, 17p13, 22q11), and three IgH translocations [t(4,14), t(11,14), t(14,16)] were available. Deletion of 8p21 and 13q14 were shown associated with HBV positivity within hepatocellular carcinoma in other investigations. In the present evaluation, the odds ratio for loss of 8p21 was significantly elevated (OR = 2.74, 95% CL = 1.36–5.50, P = 0.0048) and for loss of 13q14 non‐significantly increased (OR = 1.40, 95% CL = 0.74–2.65) in anti‐HBc positive patients. The results provide further support for a role of HBV infection in the pathogenesis of MM.     

Visceral leishmaniasis in a lung transplant recipient: usefulness of highly sensitive real‐time polymerase chain reaction for preemptive diagnosis

We report the case of a lung transplant recipient in whom the diagnosis of visceral leishmaniasis (VL) was made by detection of parasites in a peripheral blood smear when the parasite load already reached 8.9 × 10³ parasites/mL. We demonstrated that the VL diagnosis could have been done months before the development of symptoms by the use of Leishmania‐specific real‐time polymerase chain reaction (PCR), suggesting the role of preemptive PCR‐based diagnosis in transplant recipients at risk for VL.     

Significance of HTLV‐1 proviral load quantification by real‐time PCR as a surrogate marker for HTLV‐1‐infected cell count

We developed a real‐time (RT) PCR quantitative assay to measure the level of the integrated viral genome of HTLV‐1 in host peripheral blood‐mononuclear cells (PB‐MNC) from healthy carriers and patients with adult T‐cell leukemia (ATL). All of the clinical specimens were serologically and molecularly characterized by enzyme‐linked immunosorbent assay (ELISA) and Southern blot hybridization (SBH) analyses. The assay system for quantifying the proviral copy level was sensitive, accurate, and reproducible over a wide range of density from 100 to 0.1% with a coefficient of variation (%) of 4.5 to 9.6. The proviral load of the healthy carriers and patients with ATL was 301 ± 339 copies per 10⁴ MNC (3 ± 3.4%) on average and varied depending on the ATL cell number and the SBH band‐status of single or multiple bands. In ATL cases with multiple bands detected by SBH analysis, their ATL cells were shown to harbor multiple copies within one ATL cell, so that the corrected copy number interpolated by the band number in SBH was closely equivalent to the expected ATL cell number in PB, corresponding to the virus‐infected cell burden. The proviral load in healthy carriers ranged from 0.1 to 15% of PB‐MNC, and, in combination with the fraction (%) of ATL‐like flower cells defined by PB smear morphology, enabled carriers to be subgrouped into three categories. This result indicates that the detection of proviral load by(RT) PCR is sufficient and relevant to monitor the infected cell number in the PB and to evaluate the HTLV‐1 pathologic status.     

Role of IFN‐λs,IFN‐λs related genes and the DEPDC5 gene in Hepatitis B virus‐related liver disease

Recent studies have associated genetic variation near the interleukin 28B (IL28B/IFN‐λ3) gene with natural clearance of the hepatitis C virus (HCV) infection, and a common variant in the DEP domain containing 5 (DEPDC5) locus on chromosome 22 has been shown to affect susceptibility to hepatocellular carcinoma (HCC) in Japanese individuals with chronic HCV infection. This study was conducted to determine whether polymorphisms near or in interferon‐lambda (IFN‐λs) genes and their receptor genes such as interleukin 28 receptor, alpha (IL28RA) and interleukin 10 receptor, beta (IL10RB) as well as p21_activated kinases 4 (PAK4) and iron/zinc purple acid phosphatase‐like protein (PAPL), which are locate upstream of IFN‐λs, and lastly the DEPDC5 gene are associated with hepatitis B virus‐related liver disease in Han Chinese. The study subjects included 507 normal healthy controls, 350 individuals with natural clearance of HBV and 792 HBV‐infected patients. The patients were categorized into 157 inactive carriers (Case I), 216 active carriers (Case II), 111 cirrhotics (Case III) and 308 HCC patients (Case IV) subgroups. Seven single nucleotide polymorphisms (SNPs) were genotyped using the Matrix‐assisted Laser Desorption/Ionisation mass spectrometric (MALDI‐TOF MS) SNP genotyping assay. Rs423058 upstream of PAPL, rs2834167 in IL10RB and rs1012068 in DEPDC5 were associated with chronic HBV status, HBV natural clearance and the presence of HCC (P = 0.0004–0.024), respectively. PAPL, IL10RB and DEPDC5 polymorphisms have an impact on progression of HBV‐related liver disease. However, IFN‐λs genes as a tool to differentiate between different clinical courses of HBV infection were not useful in the Han Chinese population.     

Epstein‐Barr virus load in the peripheral blood of patients with rheumatoid arthritis: Accurate quantification using real‐time polymerase chain reaction

Objective

To determine whether patients with rheumatoid arthritis (RA) have elevated Epstein‐Barr virus (EBV) load in their peripheral blood mononuclear cells (PBMCs) and whether it is correlated with the HLA–DR genes they express, we developed an accurate EBV DNA quantitative assay using real‐time polymerase chain reaction (PCR) with fluorescent probes.

Methods

We studied the EBV DNA load in the PBMCs of 84 patients with RA, 69 normal controls, and 22 patients with rheumatic conditions other than RA. A 214‐bp segment from the long internal repeat of EBV was amplified from 500 ng of PBMC DNA (150,000 cells) and quantified by real‐time PCR with fluorescent probes.

Results

We demonstrated that in patients with RA, the EBV DNA load in PBMCs is increased almost 10‐fold compared with that in normal controls. The EBV load is stable over time and is not obviously influenced by disease‐modifying antirheumatic drugs or HLA–DR.

Conclusion

Patients with RA have elevated EBV load in their peripheral blood.