A complementary tool for management of disseminated Histoplasma capsulatum var. capsulatum infections in AIDS patients

In South America, disseminated histoplasmosis due to Histoplasma capsulatum var. capsulatum (H. capsulatum), is a severe and frequent opportunistic infection in AIDS patients. In areas outside the USA where specific-Histoplasma antigen detection is not available, the diagnosis is difficult. With the galactomannan antigen (GM) detection, a test commonly used for invasive aspergillosis diagnosis, there is a cross-reactivity with H. capsulatum that can be helpful for the diagnosis of histoplasmosis. The aim of this study was to evaluate the GM detection for the diagnosis of disseminated histoplasmosis in AIDS patients. The performance of the GM detection was evaluated with serum collected in French Guiana where H. capsulatum is highly endemic. Sera from AIDS patients with disseminated histoplasmosis occurring from 2002 to 2009 and from control HIV-positive patients without histoplasmosis were tested with the GM detection and Histoplasma-specific antibody detection (IEP). In 39 AIDS patients with proven disseminated histoplasmosis, the sensitivity of the Histoplasma IEP was only 35.9% and was linked to the TCD4+ lymphocyte level. For the GM detection, the sensitivity (Se) was 76.9% and specificity (Sp) was 100% with the recommended threshold for aspergillosis diagnosis (0.5). The test was more efficient with a threshold of 0.4 (Se: 0.82 [95% CI: 0.66–0.92], Sp: 1.00 [95% CI: 0.86–1.00], LR+: >10, LR−: 0.18). This study confirms that the GM detection can be a surrogate marker for the diagnosis of disseminated histoplasmosis in AIDS patients in endemic areas where Histoplasma EIA is not available.     

PCR detection of JC virus DNA in brain tissue from patients with and without progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system, which is thought to be a result of the reactivation of JC virus (JCV), a human polyomavirus. The disease occurs in individuals with immunosuppression and in recent years there has been an increase in PML cases due to AIDS. A nested polymerase chain reaction (n-PCR) was employed to detect JCV and BK virus (BKV) DNA in brain tissue collected postmortem from 28 AIDS patients with PML and from 13 patients without PML, but with other diagnoses, including solid tumors, Alzheimer's disease, thromboembolism, myocardial infarction and acute cerebrovascular diseases. All 28 brain specimens from the patients with PML were positive for JCV DNA when tested by n-PCR and three of the latter were also positive for BKV DNA. These results were confirmed by an enzyme restriction analysis and a DNA hybridization assay. Interestingly, in this study, JCV DNA was also found in 6 brain tissue specimens from 4 subjects with diseases unrelated to PML or AIDS. All the brain specimens from the control group were negative for BKV DNA. The results confirm that the n-PCR is a useful tool for PML diagnosis. The presence of JCV DNA in the brain tissue of patients without PML is particularly important since it indicates that JCV could be latent in the brains of immunocompetent individuals. Moreover, detection of simultaneous presence of JCV and BKV in the brain tissue of the patients with PML demonstrates that BKV may also infect the human brain without causing any apparent neurological disease. © Wiley-Liss, Inc.     

Application of PCR amplification of DNA from paraffin embedded tissue sections to linkage analysis in familial retinoblastoma.

A family segregating for the retinoblastoma predisposition gene has been analysed using the polymerase chain reaction to exclude their son as being an affected gene carrier. The unusual feature of this family is that the affected child, who would ordinarily have been used to establish phase in a linkage study, died as a result of developing a second tumour some years ago. The only tissue available from this child was a paraffin embedded, formalin fixed histopathological specimen from the second tumour. It was possible to isolate DNA from this tissue and amplify the DNA flanking two polymorphic restriction enzyme sites to establish alleles which cosegregated with tumour predisposition. Archival material can now be used to offer families such as this prenatal screening to provide informed genetic counselling.     

Identification of Trichophyton rubrum by nested PCR analysis from paraffin embedded specimen in trichophytia profunda acuta of the glabrous skin.

Trichophytia profunda acuta of the glabrous skin (TPAGS) arose in a 67-year-old Japanese man. The patient presented indurated erythematous plaques and nodules on his left forearm. Direct microscopic examination of the scale in KOH preparation was negative for fungal elements, and culture for dermatophytes was also negative. Although fungal infection could not be proven in hematoxillin-eosin stained sections, deep-cut sections of the biopsied skin lesion with PAS stain revealed the ectothrix presence of fungal elements. Nested PCR was done with Trichophyton specific primers directed to internal transcribed spacer gene 1 (ITS1), using template DNA obtained from formalin fixed, paraffin embedded skin sections. A single band corresponding to T. rubrum was obtained, and the etiological agent was thus identified. KOH tests and cultures may often turn out unsuccessful, perhaps reflecting the hair follicle dominant fungus growth in TPAGS. Although these tests are most important for diagnosis of TPAGS, nested PCR using paraffin embedded skin sections may be an alternative method to identify the etiological agent.     

DNA analysis of cystic fibrosis in Brazil by direct PCR amplification from Guthrie cards

A 3 bp deletion of condon 508 (phenylalanine) of the cystic fibrosis (CF) gene constitutes the mutation of most CF chromosomes. The frequency of this mutation (referred to as ΔF508), varies considerably between populations, ranging form 26% of the CF mutations in Turkey to 88% in Denmark. To determine the frequency of the ΔF508 mutation in Brazilian Caucasoid CF patients, we used direct polymerase chain reacion (PCR) amplification of DNA obtained from dried blood spots on Guthrie cards, followed by ethidium bromide staining of gels. Although the overall frequency of the ΔF508 mutation was 47% of 380 CF chromosomes from Brazilian Caucasoids born in five different states, significant inter-state differences were observed, ranging from a ΔF508 frequency of 27% to 53%. While our method could be used to screen patients and their relatives for carrier testing and prenatal diagnosis, the efficacy of screening only for the ΔF508 mutation would be low, and would vary from state to state. Screening for a panel of local mutations will be needed to increase the mutation detection rate and optimize genetic counseling. © 1993 Wiley-Liss, Inc.     

Minimal effect of delayed sample processing on results of quantitative PCR for cytomegalovirus DNA in leukocytes compared to results of an antigenemia assay.

Quantitative cytomegalovirus antigenemia and DNAemia were determined in peripheral leukocytes of 25 patients stored for up to 72 h at room temperature (RT) and 4 degrees C before processing. Numbers of antigen-positive cells significantly decreased with time. The decline was greater at RT than at 4 degrees C. In contrast, no significant alterations in DNAemia occurred.     

PCR detection of amplified 132 bp repeats in Marek's disease virus type 1 (MDV-1) DNA can serve as an indicator for critical genomic rearrangement leading to the attenuation of virus virulence

A radioactive PCR test was developed that amplified the very virulent Marek's disease virus-1 (vvMDV-1) DNA sequence containing the 132 bp repeats. In apathogenic MDV-1 (CVI 988, Rispens), amplified DNA bands containing multiple copies of 132 bp repeats were identified. In the present study this PCR technique was used to monitor the passage level of vvMDV-1 in chicken embryo fibroblasts (CEF) in which the number of tandem 132 bp repeats was increased. It was found that at passage level 32 of vvMDV-1-B isolate, the 132 bp tandem repeat was already markedly amplified and its pattern resembled that of the MDV-1 (CVI 988, Rispens) vaccine virus DNA. In the vvMDV-1Z strain, amplification of the 132 bp repeat was not detectable at a similar passage level. The PCR test demonstrated that the apathogenic MDV-1 Md11/75c virus developed by extensive in vitro passaging has amplified 132 bp DNA repeats similar to those of the commercial vaccine virus (CVI 988, Rispense). It was also found that the pattern of viral RNA from infected cells detectable by Northern blot hybridization was markedly changed from a 2.4 kb RNA species in cells infected with vvMDV-1 viruses, to four RNA species (ranging from 2.2 to 4.4 kb) in cells infected with passage 32 of MDV-1-B strain, to a very large number of undefined RNA species synthesized in cells infected with attenuated MDV-1 viruses (CVI 988, Rispens and Md 11/75c).     

Detection of H3F3A p.G35W and p.G35R in giant cell tumor of bone by Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR)

Giant Cell Tumor (GCT) represents about 20% of benign bone tumors, is locally aggressive although malignant transformation is extremely rare, <1% of cases but 2–3% give pulmonary metastasis. Age at onset is between 20 and 40 years with a slight predominance for the female gender.GCT is characterized by specific mutations in H3F3A gene encoding the protein histone 3.3. The study of these mutations is important for the differential diagnosis with giant cell rich sarcomas, chondroblastoma and aneurysmal bone cyst.To identify the most frequent H3F3A mutations we developed a novel allele specific Real Time Polymerase Chain Reaction method, based on Allele Specific Locked Nucleic Acid (ASLNAqPCR) that is here described. Molecular analyses were performed on 20 GCT and 2 osteosarcoma arising on a previous GCT. All cases were verified by Sanger sequencing. We demonstrated that ASLNAqPCR is a quick, sensitive and reliable method to identify mutations of the H3F3A gene, in giant cell tumor of bone, to support diagnosis in morphologically ambiguous cases.