Tag: Southern blot

Detection of Mycoplasma genus and Mycoplasma fermentans by PCR in patients with Chronic Fatigue Syndrome

Abstract:

Mycoplasma fermentans and other Mycoplasma species are colonizers of human mucosal surfaces and may be associated with human immunodeficiency virus infection. While many infectious agents have been described in different percentages of patients with Chronic Fatigue Syndrome (CFS), little is known about the prevalence of mycoplasmas and especially M. fermentans in CFS patients.

A polymerase chain reaction (PCR)-based assay was used to detect Mycoplasma genus and M. fermentans genomes in peripheral blood mononuclear cells (PBMC) of CFS patients. Blood was collected from 100 patients with CFS and 50 control subjects. The amplified products of 717 bp of Mycoplasma genus, and 206 bp of M. fermentans were detected in DNA purified from blood samples in 52% and 34% of CFS samples, respectively. In contrast, these genomes were found in only 14% and 8% of healthy control subjects respectively (P < 0.0001).

All samples were confirmed by Southern blot with a specific probe based on internal sequences of the expected amplification product. Several samples, which were positive for Mycoplasma genus, were negative for M. fermentans indicating that other Mycoplasma species are involved. A quantitative PCR was developed to determine the number of M. fermentans genome copies present in 1 microg of DNA for controls and CFS patients.

Mycoplasma copy numbers ranging from 130 to 880 and from 264 to 2400 were detected in controls and CFS positive subjects, respectively. An enzyme immunoassay was applied for the detection of antibodies against p29 surface lipoprotein of M. fermentans to determine the relationship between M. fermentans genome copy numbers and antibody levels. Individuals with high genome copy numbers exhibited higher IgG and IgM antibodies against M. fermentans specific peptides. Isolation of this organism by culture from clinical specimens is needed in order to demonstrate specificity of signal detected by PCR in this study.

 

Source: Vojdani A, Choppa PC, Tagle C, Andrin R, Samimi B, Lapp CW. Detection of Mycoplasma genus and Mycoplasma fermentans by PCR in patients with Chronic Fatigue Syndrome. FEMS Immunol Med Microbiol. 1998 Dec;22(4):355-65. http://femsim.oxfordjournals.org/content/22/4/355.long (Full article)

 

High prevalence without reactivation of herpes virus 6 in subjects with chronic fatigue syndrome

Abstract:

INTRODUCTION: Chronic fatigue syndrome (CFS) is a disorder of unknown etiology. Some viruses have been associated with CFS etiology, specially herpesviruses, enteroviruses and retroviruses. Some studies suggest an association between human herpesvirus-6 (HHV-6) and CFS. In order to know if there is an active HHV-6 infection in CFS patients we studied the immunologic and virologic status of HHV-6.

MATERIALS AND METHODS: Twenty patients with CFS were studied. IgG and IgM anti HHV-6 were determined by indirect immunofluorescence assay. DNA from serum and peripheral blood mononuclear cells (PBMC) were studied by dot- and Southern-blotting and nested-PCR to detect HHV-6 DNA. HHV-6 RNA from PBMC were amplified by RT(retrotranscription)-PCR.

RESULTS: Ten patients (50%) had IgG anti-HHV-6 in serum but none had IgM anti-HHV-6. Dot-blotting of DNA from 200 microliters of serum and Southern-blotting of 10 micrograms of PBMC DNA were negative. Nested-PCR from sera were negative. Nested-PCR with 1 microgram PBMC DNA were positive in 8 out 20 (40%) and with 5 micrograms PBMC DNA were positive in 16 out of 20 (80%). No viral RNA were detected in PBMC.

CONCLUSIONS: There is a high proportion of CFS patients infected with HHV-6 but with low viral load. Results do not support HHV-6 reactivation in CFS patients.

 

Source: Cuende JI, Civeira P, Diez N, Prieto J. High prevalence without reactivation of herpes virus 6 in subjects with chronic fatigue syndrome. An Med Interna. 1997 Sep;14(9):441-4. [Article in Spanish] http://www.ncbi.nlm.nih.gov/pubmed/9453750

 

Biological and molecular characteristics of human herpesvirus 7: in vitro growth optimization and development of a syncytia inhibition test

Abstract:

Two isolates of human herpesvirus 7 (HHV-7) were recovered from phytohemagglutinin-activated peripheral blood mononuclear cells of a patient with chronic fatigue syndrome and of a healthy blood donor. A genetic polymorphism between the two isolates was detected by Southern blot analysis using a novel HHV-7 genomic clone (pVL8) as a probe. We developed optimized conditions for the in vitro propagation of HHV-7 by using enriched populations of activated CD4+ T lymphocytes derived from normal peripheral blood, resulting in the production of high-titered extracellular virus (> 10(6) cell culture infectious doses/ml). Bona fide syncytia formation was documented both in normal CD4+ T lymphocytes and in the Sup-T1 CD4+ T-cell line following infection with high-titered HHV-7. To identify neutralizing antibodies to HHV-7, a syncytia-inhibition test was developed. Variable titers of syncytia-neutralizing antibodies were detected in all the human sera tested, thus confirming the high prevalence of HHV-7 in the human population.

 

Source: Secchiero P, Berneman ZN, Gallo RC, Lusso P. Biological and molecular characteristics of human herpesvirus 7: in vitro growth optimization and development of a syncytia inhibition test. Virology. 1994 Jul;202(1):506-12. http://www.ncbi.nlm.nih.gov/pubmed/8009865

 

Lack of evidence for infection with known human and animal retroviruses in patients with chronic fatigue syndrome

Abstract:

We investigated 21 patients with chronic fatigue syndrome who were identified through the surveillance system of the Centers for Disease Control and Prevention (CDC) in Atlanta for the presence of several human and animal retroviruses. In addition, we evaluated 21 CDC employee controls matched with the patients for age (+/- 5 years), gender, and race.

The viruses tested included human T-lymphotropic viruses types I and II; human spuma retrovirus; simian T-lymphotropic virus type I; simian retroviruses types 1, 2, and 3; bovine leukemia virus; feline leukemia virus; and gibbon ape leukemia virus.

Samples of peripheral blood lymphocytes and leukocytes from patients and controls were analyzed in a blinded fashion for retroviral sequences; polymerase chain reaction (PCR) amplification assays and Southern blot hybridization to 32P-labeled internal oligoprobes were used. All PCR assays were optimized for maximal sensitivity on respective infected cell lines or plasmids, and sensitivity controls were included in each experiment.

All samples from patients and controls were negative for the tested retroviral sequences. Our data indicate that none of these retroviruses plays an etiologic role or is a cofactor in the chronic fatigue syndrome illnesses of our study population.

 

Source: Heneine W, Woods TC, Sinha SD, Khan AS, Chapman LE, Schonberger LB, Folks TM. Lack of evidence for infection with known human and animal retroviruses in patients with chronic fatigue syndrome. Clin Infect Dis. 1994 Jan;18 Suppl 1:S121-5. http://www.ncbi.nlm.nih.gov/pubmed/8148438

 

Assessment of a retrovirus sequence and other possible risk factors for the chronic fatigue syndrome in adults

Abstract:

OBJECTIVE: To assess whether the human T-lymphotropic virus type II (HTLV-II) gag gene sequence, a purportedly new laboratory marker of the chronic fatigue syndrome (CFS), and other possible risk factors for CFS, particularly those associated with retroviral transmission, are associated with well-characterized CFS.

DESIGN: Two matched case-control studies.

SETTING: The metropolitan Atlanta area.

PATIENTS: Twenty-one patients with CFS who were identified by the Centers for Disease Control and Prevention CFS surveillance system; 21 CDC employee controls (laboratory study) and 42 neighborhood controls (risk-factor study) who were matched to patients by age, race, and gender.

MEASUREMENTS: Peripheral blood lymphocytes and leukocytes were assayed for the HTLV-II gag gene sequence by polymerase chain reaction and specific Southern blot hybridization. Questionnaires elicited demographic and clinical information and a history of exposures associated with retrovirus transmission (for example, blood transfusions, sexual practices, intravenous drug use).

RESULTS: All patients were white and 86% were female. The median age at illness onset was 34 years (range, 16 to 51 years). The HTLV-II gag gene sequence was not identified in the blood of any patient or control under conditions in which the appropriate assay controls were positive. No statistical differences were observed between patients and controls in frequency of blood transfusions (10% compared with 7%), median number of sex partners before illness (3 compared with 3), bisexual or homosexual behavior (14% compared with 7%), intravenous drug use (0% compared with 0%), and other factors associated with retroviral infection.

CONCLUSIONS: The HTLV-II gag gene sequence was not a marker for CFS in this small study of well-defined patients, nor did other characteristics of the patients and controls support the hypothesis that a retrovirus, transmitted by usual modes, was a cause of CFS.

 

Source: Khan AS, Heneine WM, Chapman LE, Gary HE Jr, Woods TC, Folks TM, Schonberger LB. Assessment of a retrovirus sequence and other possible risk factors for the chronic fatigue syndrome in adults. Ann Intern Med. 1993 Feb 15;118(4):241-5. http://www.ncbi.nlm.nih.gov/pubmed/8420441

 

Search for retrovirus in the chronic fatigue syndrome

Abstract:

AIM: To examine peripheral blood and skeletal muscle from patients with chronic fatigue syndrome for exogenous retrovirus.

METHODS: Blood samples from 30 patients and muscle biopsy specimens of 15 patients were examined for retroviral sequences by DNA extraction, polymerase chain reaction (PCR), and Southern blotting hybridisation. Sera were examined for human foamy virus by western immunoblotting and indirect immunofluorescence techniques.

RESULTS: No differences between the patient and control populations was found for any of the PCR primer sets used (gag, pol, env, and tax regions of HTLV I/II). An endogenous gag band was observed in both the patient and control groups. All sera were negative for antibody to human foamy virus.

CONCLUSION: The results indicate that there is no evidence of retroviral involvement in the chronic fatigue syndrome.

 

Source: Gow JW, Simpson K, Schliephake A, Behan WM, Morrison LJ, Cavanagh H, Rethwilm A, Behan PO. Search for retrovirus in the chronic fatigue syndrome. J Clin Pathol. 1992 Dec;45(12):1058-61. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC494996/ (Full article)

 

Detection of Epstein-Barr virus with molecular hybridization techniques

Abstract:

The cord-blood transformation assay remains the standard method for detecting Epstein-Barr virus (EBV) in secretions. However, newer methods are much faster and more sensitive, although most are still regarded as research procedures. The most useful of these are Southern blot hybridization, particularly the variation that employs terminal genomic probe analysis; in situ cytohybridization; and polymerase chain reaction analyses. Use of these methods alone or in combination should disclose the infected cell type, whether the infection is productive or latent, and the presence of multiple strains of EBV. Such information may help establish whether EBV is a causal agent in chronic fatigue syndrome.

 

Source: Pagano JS. Detection of Epstein-Barr virus with molecular hybridization techniques. Rev Infect Dis. 1991 Jan-Feb;13 Suppl 1:S123-8. http://www.ncbi.nlm.nih.gov/pubmed/1850538