Rosario Cultrera

The role of stage-specific oligonucleotide primers in providing effective laboratory support for the molecular diagnosis of reactivated Toxoplasma gondii encephalitis in patients with AIDS

The switch from bradyzoites to tachyzoites is the fundamental pathogenic event that leads to Toxoplasma gondii encephalitis (TE) in patients with AIDS. Distinction between these stages is difficult, particularly when specific treatment has been started. A new approach consisting of a nested PCR (n-PCR) assay was performed on cerebrospinal fluid (CSF) specimens collected from AIDS patients with TE before or after antiparasitic therapy was initiated, to assess the efficacy of primer sets which amplify target sequences expressed on bradyzoites (SAG4 and MAG1), tachyzoites (SAG1) or both stages (B1) of T. gondii. CSF specimens were obtained from 46 patients with AIDS, of whom 27 had TE (16 first episode, 11 relapse) and 19 had other AIDS-related brain lesions (AIDS-OBL) in the absence of TE. CSF specimens from 26 HIV-negative and immunocompetent patients were also checked. All samples were tested with different primer pairs targeting the B1, SAG-1, SAG-4 and MAG-1 genes. With B1, 75% of patients with first episodes of TE were positive, compared with 36.3% of those with relapse of TE and 5.2% of those with AIDS-OLB. The SAG1 gene yielded positive values in 28.7% and 45.4% of patients with first episodes of TE or relapse of TE, respectively, and in none of the controls. With the SAG4 and MAG1 genes, 72.7% of patients with relapse of TE were detected, compared with 25% of patients with first episodes of TE and 5.2% with AIDS-OLB. None of the HIV-negative subjects showed positive PCR reactions. These results demonstrate that specific primers for the genes SAG4, MAG1 and SAG1 may be useful in AIDS patients with relapse of TE, in whom the use of PCR targeting the B1 gene may fail to detect DNA, especially when prophylaxis or treatment has been started.

Molecular evidence of Ureaplasma urealyticum and Ureaplasma parvum colonization in preterm infants during respiratory distress syndrome.

BackgroundUreaplasma urealyticum and U. parvum have been associated with respiratory diseases in premature newborns, but their role in the pathogenesis of the respiratory distress syndrome (RDS) is unclear. The aim of this study was to detect, using molecular techniques, the role of Mycoplasma spp. and Ureaplasma spp. in respiratory secretion and blood specimens of preterm newborns with or without RDS and to evaluate the prevalence of perinatal U. urealyticum or U. parvum infection. The influence of chemotherapy on the clinical course was also evaluated.MethodsTracheal aspirate or nasopharingeal fluid samples from 50 preterm babies with (24) or without RDS (26) were analysed for detection of U. urealyticum and U. parvum by culture identification assay and PCR. Sequencing analysis of amplicons allowed us to verify the specificity of methods. Clarithromycin (10 mg kg-1 twice a day) was administered in ureaplasma-positive patients who presented clinical signs of RDS.Results15/24 neonates with RDS (p < 0.001) and 4/26 without RDS were found PCR-positive for U. urealyticum or U. parvum. Culture identification assay was positive in 5/50 newborns, three of which with RDS. Sequencing analyses confirmed the specificity of these methods. Association of patent ductus arteriosus with ureaplasma colonization was more statistically significant (p = 0.0004) in patients with RDS than in those without RDS.ConclusionColonization of the lower respiratory tract by Ureaplasma spp. and particularly by U. parvum in preterm newborns was related to RDS. The routine use of molecular methods could be useful to screen candidate babies for etiologic therapy.

Cerebrospinal fluid molecular demonstration of Chlamydia pneumoniae DNA is associated to clinical and brain magnetic resonance imaging activity in a subset of patients with relapsing-remitting multiple sclerosis

To further explore the link between Chlamydia pneumoniae and multiple sclerosis (MS), we examined cerebrospinal fluid (CSF) samples from 71 patients with MS and from 72 patients suffering from other inflammatory neurological disorders (OIND) or noninflammatory neurological disorders (NIND). All samples were analysed by a touchdown nested polymerase chain reaction (n-PCR) forC. pneumoniae with primer sets which amplify target sequence genes encoding the major outer membrane protein (MOMP), the16S rRNA and the Hsp- 70 protein. A molecular study was also performed to evaluate genetic diversity among isolates of C. pneumoniae and to compare chlamydial sequences. PCR was found positive in 36.6% of total MS, in 28.1% of OIND and in 37.5% of NIND patients, without any statistical differences among the various groups examined. CSF PCR evidence of C. pneumoniae was significantly more frequent in relapsing-remitting (RR) than in secondary progressive (SP) (PB-0.001) and in primary progressive (PP)MS (PB-0.05), in clinically active than in clinically stable MS (PB-0.05) and in MRI active than in MRI inactive MS(PB-0.001). The analysis of CSF expression of each single C. pneumoniae-specific gene revealed that detectable levels of MOMP were significantly more frequent in MS patients with relapse (PB-0.05), whereas PCR positivity for MOMP and 16S rRNA genes were more represented in MS patients with clinical and MRI evidence of disease activity (PB-0.05). Similar rates for MOMP and 16S rRNA genes were detected in CSF of both MS patients and controls, whereas CSF PCR positivity for Hsp-70 gene was observed in only three active RR MS patients. Sequence analysis revealed significant homologies withC. pneumoniae compared to otherChlamydial spp. These findings confirm that theC. pneumoniae detection within the central nervous system (CNS) is not selectively restricted to MS, but accounts in a variety of neurological diseases. In addition, our results suggest that CSF C. pneumoniae-specific DNA detection can occur in a subset of MS patients with clinical and MRI active RR form in whom a C. pneumoniae brain chronic persistent infection may play a significant role in the development of disease.

Chlamydophila pneumoniae Infection and Its Role in Neurological Disorders

Chlamydophila pneumoniae is an intracellular pathogen responsible for a number of different acute and chronic infections. The recent deepening of knowledge on the biology and the use of increasingly more sensitive and specific molecular techniques has allowed demonstration of C. pneumoniae in a large number of persons suffering from different diseases including cardiovascular (atherosclerosis and stroke) and central nervous system (CNS) disorders. Despite this, many important issues remain unanswered with regard to the role that C. pneumoniae may play in initiating atheroma or in the progression of the disease. A growing body of evidence concerns the involvement of this pathogen in chronic neurological disorders and particularly in Alzheimers disease (AD) and Multiple Sclerosis (MS). Monocytes may traffic C. pneumoniae across the blood-brain-barrier, shed the organism in the CNS and induce neuroinflammation. The demonstration of C. pneumoniae by histopathological, molecular and culture techniques in the late-onset AD dementia has suggested a relationship between CNS infection with C. pneumoniae and the AD neuropathogenesis. In particular subsets of MS patients, C. pneumoniae could induce a chronic persistent brain infection acting as a cofactor in the development of the disease. The role of Chlamydia in the pathogenesis of mental or neurobehavioral disorders including schizophrenia and autism is uncertain and fragmentary and will require further confirmation.

Advanced laboratory techniques for diagnosing Toxoplasma gondii encephalitis in AIDS patients: significance of intrathecal production and comparison with PCR and ECL-western blotting

The polymerase chain reaction (PCR) for detection of cerebral spinal fluid (CSF) Toxoplasma gondii DNA was combined with the study of intrathecal antibody synthesis by antibody specific index calculation (ASI) and the detection of specific oligoclonal IgG bands (OCB) by affinity mediated immunoblotting (AMI) in 11 AIDS patients with T. gondii encephalitis (TE) and in 20 control patients with or without neurological disorders. Enhanced chemiluminescence (ECL) western-blot technique was employed to evaluate the antigenic specificity of CSF-IgG towards individual T. gondii antigens. PCR was positive in all TE patients which displayed brain-derived or blood-derived specific OCB, even when comparative ASI failed. Four TE patients had a unique anti-T. gondii OCB restricted to the CSF and a strong antibody response toward the 29 kDa band by ECL western blot. This response could be an important marker to discriminate TE from other opportunistic central nervous system (CNS) infections in the course of AIDS.

Employment of terbinafine against Pneumocystis carinii infection in rat models

The anti‐Pneumocystis carinii response of terbinafine together with that of three other compounds, trimethoprim sulphamethoxazole (TMP‐SMX), atovaquone (ATQ) and albendazole (ALB), has been investigated in immunosuppressed Sprague‐Dawley rats with established pneumocystosis. Drugs were administered orally (terbinafine in dosages of 40 and 80mg/kg per day, TMP 12·5 mg/kg per day plus SMX 62·5 mg/kg per day, ATQ 100 mg/kg per day and ALB 600 mg/kg per day) to six rat groups except one which served as a control, P. carinii pneumonia (PCP) was identified post‐mortem in nine (90%) of the control rats which exhibited a marked P. carinii burden, and mean lung weights were higher with respect to the other treatment groups. During treatment, five rats in the control group died, whereas between 11 and 13 rats in all treatment groups survived. In the terbinafine groups (40 mg and 80 mg/kg per day), a mild P, carinii infection developed in three and two rats (27·2 and 18%), respectively, and almost the same infectivity score was obtained for those treated with 40 mg and 80 mg/kg per day. Histological changes in the lungs in animals receiving terbinafine treatment were minimal. Among the remaining compounds the rate of infection was seven (58·3%) for the ALB treatment group and five (45‐4%) for the ATQ group (mean score 19‐4 ±7‐1 and 23 ± 2·1, respectively). In the TMP‐SMX treatment group, there were 13 surviving rats and P. carinii organisms were found in two (15·3%, mean infection score 8 ± 1·1).

Molecular identification and antibody testing of Chlamydophila pneumoniae in a subgroup of patients with HIV-associated dementia complex. Preliminary results.

Chlamydophila pneumoniae DNA was investigated by polymerase chain reaction (PCR), in cerebrospinal fluid (CSF) specimens from patients suffering from HIV-1-associated dementia complex (HADC). Four (17.3%) cases of C. pneumoniae infection were identified among 23 HADC individuals with DNA amplification of major outer membrane protein (MOMP) gene and 16S rRNA gene sequences. Sequence analysis revealed significant homologies with C. pneumoniae compared to Chlamydia trachomatis and Chlamydia psittaci. High mean levels of CSF specific anti-C. pneumoniae antibodies and C. pneumoniae antibody specific index values significantly elevated were also found by enzyme-linked immunosorbent assay (ELISA) in these patients. The results suggest a hypothetical role of C. pneumoniae in the pathogenesis or progression of HADC.

Evidence of cerebrospinal fluid free kappa light chains in AIDS patients with Toxoplasma gondii encephalitis

Cerebrospinal fluid (CSF) free light chains of kappa or lambda (FLC kappa/lambda) type were investigated by affinity mediated blotting technique (AMI) and ELISA in 28 patients of which nine with AIDS and Toxoplasma gondii encephalitis (AIDS, TE), 11 with AIDS with or without other CNS AIDS-related opportunistic infections (non-TE AIDS) and eight control patients with or without inflammatory neurological disorders (control group). CSF restricted oligoclonal FLC bands either of k or lambda isotype or both were found by AMI in 18 (90%) out of 20 AIDS patients, while a CSF pattern predominantly characterized by FkappaLC rather than FlambdaLC was observed in eight (88.8%) out of nine TE patients. No FLC components were detected in the matched sera of TE or non-TE AIDS patients or in the CSF and sera from control group. The anti-parasite-specific FkappaLC CSF/serum mean levels and the T. gondii-specific FkappaLC index values were found by ELISA to be significantly more elevated in TE patients when compared to non-TE AIDS or control group. These findings suggest that the increased production of T. gondii-specific FkappaLC could provide insights into pathogenesis of reactivated TE in immunocompromised patients and may have important diagnostic usefulness.

Intrathecal production of Chlamydia pneumoniae-specific high-affinity antibodies is significantly associated to a subset of multiple sclerosis patients with progressive forms

The purpose of this study was to provide further insight into the effective relevance of the association between Chlamydia pneumoniae and MS. We evaluated by ELISA technique cerebrospinal fluid (CSF) and serum levels of anti-C. pneumoniae IgG in 46 relapsing-remitting (RR), 14 secondary progressive (SP) and 11 primary progressive (PP) MS patients grouped according to clinical and Magnetic Resonance Imaging (MRI) evidence of disease activity. Fifty-one patients with other inflammatory neurological disorders (OIND) and 52 with non-inflammatory neurological disorders (NIND) were used as controls. A C. pneumoniae-specific intrathecal IgG synthesis as detected by the relative specific index was present in a small proportion of MS (17%), OIND (22%) and NIND (2%) patients and was significantly more frequent in MS and in OIND than in NIND (p<0.001) and in SP and PP MS than in RR MS patients (p<0.02). Among the patients with C. pneumoniae-specific intratecally produced antibodies, CSF high-affinity anti-C. pneumoniae IgG were found in the majority of SP or PP MS, occasionally in OIND, but not in RR MS and NIND patients. These findings confirm that the presence of a humoral immune response to C. pneumoniae within the central nervous system (CNS) is not selectively restricted to MS, but is shared by several inflammatory neurological conditions. In addition, our results suggest that an intrathecal production of C. pneumoniae-specific high-affinity IgG can occur in a subset of patients with MS progressive forms in which a C. pneumoniae brain chronic persistent infection may play an important pathogenetic role.

Investigation of the simian polyomavirus SV40 as a potential causative agent of human neurological disorders in AIDS patients.

Neurological diseases and a variety of neoplasms frequently occur in AIDS patients. Human JC and BK polyomaviruses have been associated with neurological disorders in such patients. SV40 polyomavirus sequences have been detected in human brain tumours, other neoplasms and normal tissues. JCV, BKV and SV40 DNA sequences were investigated in cerebrospinal fluid (CSF) samples from 12 AIDS patients affected by different neurological disorders, by PCR assay and filter hybridisation with specific internal oligoprobes, and DNA sequencing. Three of the 12 CSF samples were positive for JCV (one sample) or SV40 (one) DNA, or both (one). No sample was positive for BKV DNA. JCV- and SV40-specific genomic regions were confirmed by DNA sequencing. CSF samples from the two patients diagnosed clinically as having progressive multifocal leukoencephalopathy (PML) contained either JCV (one sample) or SV40 (one) DNA. The CSF found to contain both JCV and SV40 DNA originated from a patient with a cerebral mass lesion of unknown aetiology. These results suggest that SV40 may be involved in the aetiology of PML in AIDS patients, and raise the possibility that SV40 and JCV may act synergically in vivo to enhance their pathogenicity.