Determination of prevalence and risk factors of infection with Babesia in small ruminants from Greece by polymerase chain reaction amplification.

A total of 124 blood samples were collected from 92 sheep and 32 goats from 21 randomly selected herds located in two regions of Greece. Data on the characteristics of the animals (species, gender, age, tick burden, presence of haemoglobinuria, prior treatment for babesiosis) and the herd (location, size, species of animals, dogs associated with the herds, tick burden of dogs associated with the herds) were collected through questionnaires. Nineteen animals (15%) produced the DNA fragment specific for Babesia of which 16 were sheep and three were goats. Nucleotide sequence of PCR products revealed 100% homology with Babesia ovis 18S rRNA gene. Nine farms (43%) were found positive for B. ovis. The percentage of positive animals in each farm varied between 10 and 61%. The relative risk of the presence of ticks in sheep and goats (p<0.01) and farm dogs (p<0.01) for PCR-positive results for B. ovis in sheep and goats was found 6.63 and 4.14, respectively.

Human papilloma virus (HPV) is possibly involved in laryngeal but not in lung carcinogenesis.

Data on human papilloma virus (HPV) involvement in preneoplastic and neoplastic lesions of the larynx and lung are limited and conflicting. The presence of HPV was investigated in a series of laryngeal specimens and non-small cell lung carcinomas (NSCLCs). The laryngeal samples (154) comprised 14 cases with hyperplasia without dysplasia, 49 with dysplasia, and 91 squamous cell carcinomas (SqCCs). The NSCLCs included 31 SqCCs, 32 adenocarcinomas, and 5 undifferentiated large cell carcinomas. Furthermore, we examined, for HPV DNA sequences, 14 bronchial metaplastic squamous lesions located next to cancerous areas. We used a sensitive nested polymerase chain reaction assay (NPCR), dot blotting, and in situ hybridization. The findings were correlated with clinicopathologic features of the patients. In the laryngeal specimens, NPCR analysis showed HPV DNA in 20 (13%) of the 154 specimens. Notably, 19 of 20 HPV-positive cases were carcinomas and only one was a mild dysplastic lesion. Typing of the carcinomas showed single HPV 6, 16, 18, and 33 infection in 1 (1.1%), 12 (13.2%), 2 (2.2%), and 1 (1.1%) samples, respectively, and HPV 6/33, 16/33, and 6/18 coinfection in three carcinomas. In situ hybridization findings were in agreement with PCR results, with the exception of two cases in which HPV 18 DNA was detected only by PCR. HPV was more frequently observed in heavy smokers than in patients with low daily cigarette consumption and nonsmokers (P = .03). There was no correlation between virus infection and gender, grade, and lymph node status of the carcinomas. None of the NSCLCs or adjacent metaplastic squamous epithelium contained HPV DNA sequences. The presented data suggest a contributory role of HPV in late stages of laryngeal carcinogenesis, because all premalignant lesions were negative but one. This study does not support a potential role of HPV in the development of NSCLCs.

Sensitive differential detection of genetically related mycobacterial pathogens in archival material.

A polymerase chain reaction (PCR) assay targeted to the immunogenic protein MPB64 gene was used to detect members of the Mycobacterium tuberculosis complex, and an outward-primed PCR (OPPCR) designed on the IS6110 element allowed differentiation between Mycobacterium bovis and Mycobacterium tuberculosis. Additionally, the amplification of IS1110 and 16S ribosomal RNA sequences combined with a dot blotting assay were able to differentially detect Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium paratuberculosis. The validity of the experimental procedure was tested on reference material and formalin-fixed paraffin-embedded samples from patients with tuberculosis, sarcoidosis, or Crohn disease. We demonstrated mycobacterial DNA in 59 of 75 cases with histologic lesions typical of tuberculosis; we detected M tuberculosis and M paratuberculosis in 6 of 25 sarcoidosis cases and in 7 of 20 Crohn disease specimens, respectively. The proposed diagnostic procedure is directly applicable to archival material and allows differentiation of genetically related mycobacterial pathogens in more detail than other molecular methods. It provides a tool for the diagnostic study of tuberculosis, sarcoidosis, and Crohn disease.

Experimental inoculation of laboratory animals with samples collected from sarcoidal patients and molecular diagnostic evaluation of the results.

Studies on the implication of mycobacteria in the pathogenesis of sarcoidosis have generated conflicting results. In an attempt to further elucidate the etiology of the disease, we obtained broncho-alveolar lavage (BAL) samples from sarcoidal patients, which were subsequently used for intra-tracheal inoculation of a group of rabbits. Patients were characterized as sarcoidal on the grounds of clinical, radiographic, histological and microbiological testing. Four months following inoculation, lung and alveolar lymph node specimens were collected from the animals and were examined by means of histology and microbiology, as well as by a polymerase chain reaction (PCR) assay, targeted to DNA sequences of the Mycobacterium tuberculosis and Mycobacterium avium complexes. All of the twenty five BAL-inoculated rabbits revealed evidence of lobar pneumonia, with thirteen developing lesions of non-caseous granulomatous inflammation, similar to those observed in sarcoidal patients. Microbiological cultivation of lung and alveolar lymph node material, Zihl-Neelsen staining of corresponding tissue sections and PCR analysis of extracted DNA yielded no evidence of mycobacterial infection. Identical processing of biopsies originating from the martyrs, formerly inoculated with drinking water or disinfected BAL, revealed no pathological signs. Our findings suggest that BAL samples from patients with sarcoidosis may carry an agent that produces a disease characterized by similar histological lesions in rabbits. However, culture, and PCR, could not identify this agent as a member of Mycobacterium tuberculosis, or Mycobacterium avium complexes.

Multiplex PCR assay for the detection of mycobacterial DNA sequences directly from sputum.

Time consuming classical diagnostic tests and the increasing incidence of tuberculosis epidemics have rendered the need for new more sensitive diagnostic tools, urgent. This study explored the possibility of a direct and rapid method for the identification and characterization of pathogenic typical and atypical species of mycobacteria from sputum, based on a multiplex PCR assay. Gene bank search on the mycobacterial genome revealed specific sequences that fulfilled the above set criteria. Two pairs of primers were used to amplify a 243 bp fragment of the gene encoding the immunogenic protein MPB 64 and a 133 bp fragment of the gene encoding the 65-KDa mycobacterial antigen. The first pair of primers was selected among others, to detect specifically bacteria of the M. tuberculosis complex, whereas the second, to detect in addition to the latter, those of the M. avium-intracellular complex. Our mutiplex PCR assay, detected and identified correctly, all the mycobacterium tuberculosis and M. avium-intracellulare complex strains provided on pure culture as controls with a sensitivity of 10(-3) colony forming units. Furthermore, by performing our assay on 55 sputum samples from patients with positive culture and Ziehl-Neelsen staining, we identified mycobacterial DNA sequences in all--39 samples with M. tuberculosis complex and 16 with M. avium-intracellulare complex. Out of 300 sputum specimens from patients with clinical evidence of tuberculosis, 149 were positive by our method (95 M. tuberculosis and 54 M. avium-intracellulare complex) whereas 157 samples (95 M. tuberculosis complex, 59 M. avium-intracellulare complex, 1 M. xenopi, and 2 that could not be positively identified) were culture positive and only 95 Ziehl Neelsen positive. These findings suggest that the method described can be applied on sputum, and can identify in one step strains of the M. tuberculosis and M. avium-intracellulare complex and has effectivness comparable to culture methodologies.

Multiplex polymerase chain reaction for the detection of mycobacterial DNA in cases of tuberculosis and sarcoidosis.

The aims of the present study were: (1) to design a sensitive and specific polymerase chain reaction-based method that would allow detection of most common human typical and atypical mycobacterial strains and (2) to apply the method on formalin-fixed paraffin-embedded (FFPE) tissue and sputum samples from patients with clinicopathological evidence of tuberculosis and sarcoidosis. Three sets of primers were selected. The first detects specifically members of the Mycobacterium tuberculosis (M. tuberculosis) complex, amplifying a 243 bp fragment of the gene encoding the immunogenic protein MPB 64, whereas the second traces members of the Mycobacterium avium (M. avium) complex producing a 91 bp fragment of the IS1110 element. The third pair of primers is specific for slow-growing mycobacteria, amplifying a 383 bp region of the 65 kDa mycobacterial antigen gene. Our multiplex polymerase chain reaction assay identified mycobacterial DNA of 10(-3) colony-forming units (CFU)/mL from sputum samples, 10(-5) CFU/mL from FFPE tissue samples and 10(-6) CFU/mL from pure broth cultures. By performing the method on 75 FFPE tissue samples with histological and clinical evidence of tuberculosis and 300 sputum specimens from patients suspected of tuberculosis, we found 38 M. tuberculosis complex, 7 M. avium complex, and 14 slow-growing mycobacteria positive samples in the first case and in the second we found 95 M. tuberculosis complex, 21 M. avium complex, and 35 slow-growing mycobacteria positive samples. The sensitivity of the assay was significantly higher than that of Ziehl-Neelsen and in some cases higher than culture, especially when applied on atypical mycobacteria. In addition, 25 cases histologically and clinically characterized as sarcoidosis were investigated for mycobacterial DNA sequences and in nine of these, DNA corresponding to M. tuberculosis complex was detected. The method described can be applied directly on FFPE and sputum samples and allows not only the detection of mycobacterial DNA, but also an assessment concerning the species involved.

Effects of p53 mutants derived from lung carcinomas on the p53-responsive element (p53RE) of the MDM2 gene.

The present study represents a continuation of previous works in which we observed that lung carcinomas co-expressing MDM2 protein and p53 mutants (mt p53) exhibited more aggressive behaviour. In the above studies, we suggested a 'gain of function' mechanism of mt p53 proteins based on the fact that the MDM2 gene possesses a p53-responsive element (MDM2-p53RE). In this study, to prove our hypothesis, we selected 12 cases from a series of 51 bronchogenic carcinomas. In these 12 cases, we examined the ability of the expressed mt p53 to bind the MDM2-p53RE and correlated the findings with MDM2 expression. Furthermore, we constructed four of these p53 mutants and studied their transactivation properties by co-transfecting them with a reporter plasmid carrying MDM2-p53RE in the p53 null non-small-cell lung carcinoma cell line (NSCLC) H1299. We observed mutant p53 protein DNA-binding activity, which depended on the nature and the position of the amino acid substitution. The fact that the cases with DNA-binding activity were accompanied with MDM2 protein isoforms' overexpression is indicative of a 'gain of function' phenotype. This hypothesis was enforced by the findings of the transfection experiments, which revealed that certain p53 mutants enhanced the expression of the luciferase reporter gene either directly or indirectly via a dominant positive effect on the wild-type p53. In conclusion, this work is one first attempt to examine if the deregulation of the p53/MDM2 autoregulatory feedback loop is due to novel properties of certain p53 mutants in the specific environment of a subset of bronchogenic carcinomas.