Diagnosis of childhood BK virus cystitis by electron microscopy and PCR.

Abstract

Methods Cytological smears were prepared from urinary sediment obtained on the third, fifth, and 15th days. The smears were fixed in 95% ethanol and stained by the Papanicolaou method.The third day urinary sediment was examined electron microscopically. The cell pellet was fixed with 2-5% glutaraldehyde and 1% osmic acid, dehydrated with graded alcohol, and embedded in Polybed 812. Ultrathin sections were made, stained with uranyl acetate and lead citrate, and observed with an electron microscope (H-7000, Hitachi, Japan).About 200-300 abnormal urothelial cells on a glass slide prepared by the Papanicolaou method were used for DNA extraction. The Papanicolaou stained smear was immersed in xylene in a 60°C incubator for one day, after which the cover glass was detached. Xylene was removed with alcohol. After washing with distilled water, digestion of the protein of the urothelial cells was performed on the glass slide in 300 ,ul proteinase K solution (200,ug/ml) at 37°C for 18 hours. The DNA was then extracted with phenol. Extractions with chloroform were successively repeated three times and the phenol was eliminated. Next, the RNA in the sample was digested with 100 ug/ml RNase at 37°C for 15 minutes and the DNA was again extracted with phenol. Extractions with chloroform were then repeated three times and the RNase was completely eliminated. The DNA sample was finally precipitated with ethanol and dissolved in 50 pul of 10 mM TRIS-HCl buffer (pH 7 5) containing 1 mM EDTA. The sample was frozen and thawed several times before PCR to destroy inhibitors of Taq polymerase in the urne.7The sample DNA, the sample DNA diluted three times with phosphate buffered saline (PBS), DNAs of BK and JC viruses used as positive controls and human placental DNA used as a negative control, and PBS without DNA were prepared for amplification by polymerase chain reaction (PCR). The control BK virus DNA used had been extracted from the urine of a patient with a urinary bladder carcinoma and was found to be positive for BK virus using PCR. The JC virus DNA was extracted from the brain tis-773 on 11 May 2018 by guest. Protected by copyright.

A prospective study of BK-virus-associated haemorrhagic cystitis in paediatric patients undergoing allogeneic haematopoietic stem cell transplantation

“…[1][2][3][4] BK virus, a human polyoma virus (HuPyV), is usually acquired during infancy as an asymptomatic infection, although it has been associated with urinary tract diseases by some authors. 5,6 The primary infection results in viral latency in renal tubular epithelial and urothelial cells, but asymptomatic BK viruria has been found in 5% to 460% of healthy individuals. 7,8 In recent years, BK virus reactivation has been associated with the occurrence of late-onset HC after HSCT, [9][10][11] although other concurrent factors seem to play a role such as acute GVHD, 12,13 an unrelated donor 14 and myeloablative conditioning regimen.…”

Evaluation of Renal Biopsies for BK Virus Nephropathy in Non-Renal Transplant Immunosuppressed Patients

“…The first infection is asymptomatic or characterized with a mild respiratory disease. After the first infection, the virus becomes latent in the uroepithelium (transepithelium, renal tubular epithelium and parietal epithelium of Bowman's capsule), lymphoid tissue and many different tissues (cells of salivary glands, leukocytes in peripheral blood, squamous cells of the cervix and glandular epithelial cells of the prostate); 70-90% of healthy individuals in the general population have antibodies against BKV (3)(4)(5)(6)(7)(8).…”

Diagnóstico de vírus por microscopia eletrônica em urina de pacientes com hematúrias/cistite hemorrágica após transplante de medula óssea: associação com aspectos clínicos

“…BK e JC são algumas vezes detectados na urina de mulheres grávidas (21) e idosos, mas freqüentemente detectados em imunossuprimidos, tais como transplantados renais e de medula óssea (12,14,51,59,63,77) . Há raros relatos de cistite em criança causada por BK (63) . No TMO, a excreção urinária positiva para BK e JC tem ocorrido exclusivamente em pacientes soropositivos, indicando a probabilidade de reativação da infecção (5) .…”

“…A importância da utilização desta técnica de ME em corte ultrafino para detecção de vírus na urina já foi anteriormente demonstrada (3,24,32,36,37,63) , mas utilizada em pequenas casuísticas de pacientes submetidos a TMO, e apenas como comprobatório quanto à presença de vírus, complementando a outras técnicas empregadas.…”

“…Assim, utilizando a técnica de ME, encontramos positividade para vírus na urina 55,6% dos pacientes, com Questiona-se se os vírus representariam apenas um co-fator associado à DECH (8,11,20,25) , uma vez que a H/CH ocorreria em maior número nos transplantes alogênicos. A reativação do poliomavírus é registrada em torno de 55% em imunocomprometidos (12,14,51,59,63,77) , mas também ocorre em mulheres grávidas (21) . No caso do adenovírus é relatado como raro patógeno na H/CH (0 a 5% dos pacientes) (23) (53,71) .…”