Evidence for swine and human papillomavirus in pig slurry in Italy.

AIMS: The diversity and the geographical distribution of swine papillomaviruses (PVs) are virtually unknown. The occurrence and the diversity of swine PV were therefore investigated in pig slurry collected in Italy, to contribute towards filling this gap in knowledge. METHODS AND RESULTS: Twenty-two slurry samples underwent analysis by nested PCR and DNA sequencing using published and newly designed specific primer pairs for Sus scrofa papillomavirus (SsPV) type 1 and 2 (SsPV1 and 2), along with degenerate PV-specific primers targeting the major coat protein L1 and the helicase protein E1. Overall, three samples (13·6%) were positive for SsPV1 by specific primers, and nucleotide (nt) sequences showed 99-100% nt identity with SsPV1 variant a (EF395818), while SsPV2 was not found in any sample. Using generic primers, eight samples (36·4%) were tested positive for human papillomavirus (HPV), and were characterized as follows: ß1-HPV8, ß1-HPV14, ß1-HPV206, ß2-HPV113, ß2-HPV120 and γ1-HPV173. Moreover, one unclassified γ-type was detected. CONCLUSIONS: Both swine and human PVs were detected in pig slurry in this study. The unexpected presence of HPV in pig waste could be explained as the result of an improper use of the sewage collection pits and/or with improper procedures of the operators. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports the first detection of SsPV1 in Italy, along with the first detection of HPVs in pig slurry samples in Italy, and expands our knowledge about PV diversity and geographic distribution.

Evaluation of a new plate hybridization assay for the laboratory diagnosis of imported malaria in Italy.

A new molecular diagnostic method "Malaria-IBRIDOGEN" (Amplimedical S.p.A.--Bioline Division, Turin, Italy) based on a plate-hybridization assay for the simultaneous detection and identification of human malaria parasites was evaluated in this study. A target DNA sequence of the plasmodial 18S ribosomal RNA gene was amplified by polymerase chain reaction (PCR) and hybridized in microtiter wells with five biotinylated probes each specific for Plasmodium falciparum, P. vivax, P. malariae, P. ovale and the beta-globine human gene, respectively. Compared to the nested-PCR actually used in our laboratory for the molecular diagnosis of malaria, "Malaria-IBRIDOGEN" revealed an overall sensitivity of 100% (51/51) for the four human Plasmodium species testing 100 whole blood samples from people with malaria-like symptoms and fever. Specificity was 92% (45/49) considering four discordant samples as "false positive" by "Malaria-IBRIDOGEN". The assay showed a threshold of parasite density (detection limit) of 0.07 P. falciparum parasites/microliter, 0.15-1.5 P. vivax parasites/microliter, 0.3 P. malariae parasites/microliter and 0.4 P. ovale parasites/microliter of whole blood, respectively. This assay could be successfully applied to the laboratory diagnosis of malaria as a useful aid to microscopy.

Development of a real-time PCR assay for detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for routine clinical diagnosis.

A TaqMan-based real-time PCR qualitative assay for the detection of three species of malaria parasites-Plasmodium falciparum, P. ovale, and P. vivax-was devised and evaluated using 122 whole-blood samples from patients who had traveled to areas where malaria is endemic and who presented with malaria-like symptoms and fever. The assay was compared to conventional microscopy and to an established nested-PCR assay. The specificity of the new assay was confirmed by sequencing the PCR products from all the positive samples and by the lack of cross-reactivity with Toxoplasma gondii and Leishmania infantum DNA. Real-time PCR assay showed a detection limit (analytical sensitivity) of 0.7, 4, and 1.5 parasites/ micro l for P. falciparum, P. vivax, and P. ovale, respectively. Real-time PCR, like nested PCR, brought to light errors in the species identification by microscopic examination and revealed the presence of mixed infections (P. falciparum plus P. ovale). Real-time PCR can yield results within 2 h, does not require post-PCR processing, reduces sample handling, and minimizes the risks of contamination. The assay can therefore be easily implemented in routine diagnostic malaria tests. Future studies are warranted to investigate the clinical value of this technique.

Identification of Plasmodium falciparum, P. vivax, P. ovale and P. malariae and detection of mixed infection in patients with imported malaria in Italy.

The species-specific nested-PCR previously described by Snounou and others for detecting the four parasite species that cause human malaria is evaluated in the current study testing 230 blood samples. The results are compared with those obtained by microscopy and, for 101 samples out of 230, with those previously obtained by a genus-specific PCR based method (pg-PCR) followed by species-specific Southern-blot hybridization. All blood specimens were obtained from patients (127 foreigners and 103 Italians) with a suspect clinical diagnosis of imported malaria in Italy: 76 were positive by microscopy and 83 were positive by nested-PCR. The last method also revealed 10 double infections (8 foreigners and 2 Italians) which were not identified by microscopy or by pg-PCR with species-specific Southern-blot hybridization. Fifty-four out of 83 positive samples tested by nested-PCR were submitted to genomic sequence analysis, which confirmed the presence of DNA region portion encoding the 18S rRNA corresponding to the Plasmodium species identified by nested-PCR. These results demonstrate that the nested-PCR assay surpasses microscopy and pg-PCR with species-specific Southern-blot hybridization, both in sensitivity and in diagnostic accuracy. Moreover, it is quicker because it requires no further blotting or hybridization of PCR amplification products. This method also offers a clear advantage in the detection of mixed infections, which is important not only for successful medical treatment but also for the study of malaria epidemiology. Finally, our study also highlights the value of genomic sequence analysis for validating PCR results.

Unusual paranasal sinus tumors in two patients with common nasal complaints.

Common nasal complaints are managed by both the otolaryngologist and the primary care physician. We describe the cases of two patients with nasal obstruction who were referred to us for evaluation--one with severe headache and the other with profuse epistaxis. Their histories prior to referral included long-term, common rhinologic complaints of low-grade headache and mild epistaxis. Neither patient had been referred to us until their symptoms had become severe. Our examination revealed that both patients had rare paranasal sinus pathology. One patient had a fibroxanthoma of the frontal sinus, and the other had extramedullary hematopoiesis of the maxillary sinus. Fibroxanthoma of the frontal sinus is rare, and extramedullary hematopoiesis of the maxillary sinus has not been previously reported. These two unique cases serve as a reminder that long-term common rhinologic complaints can occasionally be a sign of life-threatening pathology and require a full evaluation by an otolaryngologist.

Usefulness of genus-specific PCR and Southern blot species-specific hybridization for the detection of imported malaria cases in Italy.

A PCR method involving a genus-specific oligonucleotides set and Southern blot hybridization with four species-specific probes to P. falciparum, P. vivax, P. malariae and P. ovale was evaluated for the detection of malaria parasites in blood samples from 101 patients with clinically suspect malaria infection imported to Italy. Plasmodium falciparum was the main species detected. As determined by microscopy, 53 (52.4%) patients had malaria and of these: 40 (75.5%) were infected with P. falciparum; 7 (13.2%) with P. vivax; 1 (1.9%) with P. ovale; 3 (5.7%) with P. malariae; 1 (1.9%) with P. vivax or P. ovale; and 1 (1.9%) with P. falciparum or P. vivax. Ninety-seven out 101 blood samples were submitted to ParaSight-F test which showed a sensitivity of 94.73%, and a specificity of 93.22%, as compared to microscopy. The PCR assay using the genus-specific oligonucleotide primer set (pg-PCR) was able to detect 53 (52.4%) infections and showed a sensitivity of 100% and a specificity of 100%, when compared to microscopy. The parasite species were identified by Southern blot hybridization using species-specific probes and 40 (75.5%) samples were P. falciparum positive, 5 (9.4%) P. vivax positive, 4 (7.5%) P. ovale positive, and 2 (3.8%) P. malariae positive. When the Southern blot results were compared to those of blood-film diagnosis, we observed some disagreement. In particular, compared to Southern blot, microscopy underestimated P. ovale infection; blood film analysis recognised only 1 P. ovale sample, whereas Southern blot recognised 4 P. ovale positive samples (by microscopy, 2 of these were detected as P. vivax, 1 as P. ovale or P. vivax, and the other as P. falciparum or P. vivax). Southern blot hybridization was unable to identify one P. falciparum and one P. vivax positive case detected by microscopy. We also plan to use a reference nested-PCR assay to clarify the disagreement observed between microscopy and Southern blot hybridization.

Evaluation of OptiMAL Assay test to detect imported malaria in Italy.

This study evaluated a newly developed rapid malaria diagnostic test, OptiMAL Assay, to detect "Plasmodium falciparum malaria" and "non Plasmodium falciparum malaria" in blood samples from 139 individuals with a presumptive clinical diagnosis of imported malaria in Italy. OptiMAL Assay utilizes a dipstick coated with monoclonal antibodies against the intracellular metabolic enzyme, plasmodium Lactate Dehydrogenase (pLDH) present in and released from parasite-infected erythrocytes. Blood samples from 56 cases out of 139 were found "Plasmodium falciparum malaria" positive by microscopy; with these samples OptiMAL Assay and the ParaSight-F test, which is a kit detecting the P. falciparum histidin-rich protein 2 (HRP-2), showed an overall sensitivity of 83% and 94%, respectively, in comparison with microscopy. Parasitemia levels tested in the 56 P. falciparum positive blood samples by microscopy ranged from <0.004% to 20%. A correlation between sensitivity and parasitemia was evident and OptiMAL Assay and ParaSight-F test were more sensitive (96-100%; 100%) with samples with 0.1%-20% levels of parasitemia, while proved less sensitive (0-44%; 50-88%) with <0.004-0.01% levels of parasitemia.

More aggressive behavior of squamous cell carcinoma of the anterior tongue in young women.

OBJECTIVE: To review the combined experience from two large medical centers in treating young female patients with anterior tongue cancer to determine the clinical course of this unique subset of patients. STUDY DESIGN: Retrospective study. METHODS: Seventeen female patients less than 40 years of age (group A) and 17 older patients, both male and female, greater than 40 years of age (group B) who had treatment for invasive squamous cell carcinoma of the anterior tongue were studied. The charts were reviewed for the clinical staging, treatment, and outcome of each patient. The disease-free survival and recurrence rates were compared between the two cohorts. RESULTS: The mean disease stage between the groups was II. The survival analysis showed a significant difference between the two groups in recurrences (group A = 65%, group B = 41%; P = .02). Further, of the patients who had recurrence, the young women did so significantly earlier in their disease course than the older patients (group A = 14 mo, group B = 40 mo; P < .05). Although the survival differences did not reach statistical significance (P = .15), the power of the study was low (power = 0.26) resulting in a high-level type II error. CONCLUSION: These data suggest that young women with squamous cell carcinoma of the anterior tongue have significantly higher rates of recurrent disease and the interval to recurrence is significantly shorter than in older patients. Further investigation is warranted until a statistically significant cohort is accrued; until that time, these patients warrant an aggressive initial treatment and close surveillance for recurrence.

Search for malaria parasites by PCR and Southern blot in patients with imported malaria in Italy.

The present study evaluates the sensitivity, specificity and usefulness of a PCR method with Southern blot hybridization to detect malaria parasites in blood samples from subjects with a suspect clinical diagnosis of malaria imported to Italy. Plasmodia were detected by PCR using a genus-specific primer-set corresponding to the sequences common to P. falciparum, P. vivax, P. malariae and P. ovale, as described by Arai (Arai et al., Nucleosides Nucleotides, 1994, 13, 1363-1364) and Kimura (Kimura et al., Journal of Clinical Microbiology, 1995, 33, 2342-2346). In addition, four distinct tandemly repetitive species-specific probes, described by Kawai (Kawai et al., Analytical Biochimestry, 1993, 209, 63-69), were synthesized to specifically detect the four malaria parasites species by Southern blot hybridization. Fifteen blood samples from 12 patients (7 with malaria) were tested and the genus-specific PCR method showed a sensitivity of 100% and a specificity of 100%, when compared to microscopy, in detecting malaria parasites in the tested blood samples. Fourteen samples (nine were positive and five negative by PCR) were confirmed by Southern blot, whereas only one P. vivax positive sample was not hybridized with the species-specific probes. We conclude that this PCR method with Southern blot hybridization may be useful in detecting malaria parasites in patients with malaria imported to Italy.

Primary surgery as treatment for early squamous cell carcinoma of the tonsil.

BACKGROUND: The management of tonsil carcinoma has gradually evolved such that the literature is replete with outcome summaries of this disease treated with primary RT and chemotherapy. Recently there have been no reports of patient outcomes with primary surgical therapy. Nonsurgical treatment is warranted when tumors are unresectable or if the patient refuses surgery. Our policy has been to treat operable squamous cell carcinoma (SCCA) of the tonsil with surgery. The decision to use adjuvant therapy is based on the surgical and histologic findings. We herein report our results with this treatment protocol. METHODS: A retrospective review of 162 patients with SCCA of the tonsil was performed. Eighty-four patients were treated with surgery, which was followed by RT and/or chemotherapy if histologic signs of aggressive behavior were identified. Patients were followed 2 to 15 years after treatment. RESULTS: Of the 9 patients with stage I disease, 89% are without evidence of recurrent disease and 91% of patients with stage II tonsil cancers are also disease free. The survival rates for stage III and stage IV cancer patients are 79 and 52%, respectively. CONCLUSION: Our data suggest that patients with early tonsil cancer can be effectively treated with surgery. Surgery allows pathologic staging so that patients with advanced tumors can be treated with adjuvant therapy.

Cutaneous metastatic lung cancer: literature review and report of a tumor on the nose from a large cell undifferentiated carcinoma.

Cutaneous metastatic disease is a prognostically important diagnosis. We report the case of a 64-year-old man who had an uncommon histologic type of lung cancer--a large cell undifferentiated carcinoma, which was metastatic to the skin of the nose. The relative frequency of cutaneous metastasis is similar to that of primary cancers. Cutaneous disease as the first sign of metastasis is most often seen in cancer of the lung. However, its appearance as a large tumor on the nose, which was observed in this case, is unusual.

A pilot study evaluating the treatment of postparotidectomy sialoceles with botulinum toxin type A.

OBJECTIVE: To report our experience in treating 4 cases of recurrent siacloceles with botulinum toxin type A after partoid surgery. DESIGN: This is a prospective, nonrandomized, nonblinded pilot study describing a new use for botulinum toxin type A. SETTING: Tertiary academic medical center. PATIENTS: Four patients (2 men and 2 women) with persistent postparotidectomy sialoceles who had undergone various treatment failures were included. The diagnosis was made by fine-needle aspiration of the mass based on well-recognized cytologic features of the entity, as well as an elevated amylase level and no evidence of tumor or infection. INTERVENTIONS: Sialoceles were aspirated before local injection of botulinum toxin type A (30-50 U) subcutaneously. MAIN OUTCOME MEASURES: The patients were followed up 1 week after receiving botulinum toxin type A injection and then at monthly intervals. They were extensively questioned and examined for any evidence of side effects or recurrence. RESULTS: All patients had total resolution of sialocele or external salivary fistula within 1 month of treatment. None of the patients to date have demonstrated recurrences at 7 through 13 months, and there were no complications, particularly facial nerve weakness. CONCLUSION: Our findings suggested that botulinum toxin type A offers a highly effective, safe, and noninvasive method of treatment in postparotidectomy sialocele.

Dendritic cell sarcoma of the thyroid.

BACKGROUND: Follicular dendritic cell sarcoma (FDCS) arises from nonlymphatic antigen-presenting cells found in lymph node B-cell follicles. This extremely rare tumor, which usually arises in lymph nodes, does occur in extranodal head and neck sites such as the tonsil and soft palate. METHODS: A retrospective review of the patient followed at the University of Pittsburgh Medical Center from 1993 to the present was performed. CONCLUSIONS: This is the first reported case of an FDCS of the thyroid. A review of the literature provides useful information to aid in detection, treatment, and outcome of this unusual soft tissue malignancy.

Visual loss reversed after treatment of acute bacterial sinusitis.

Visual loss is a rare complication of acute bacterial sinusitis (ABS). Very few cases have been reported in the literature. Only two cases of visual loss reversed after treatment of ABS are found in the English-language literature. We present three cases in which significant visual loss was reversed after treatment of ABS. The experience with visual loss associated with ABS is small; therefore, no definitive statement about treatment can be made. However, on the basis of our experience, it appears that immediate surgical drainage with antibiotic therapy may be important in restoring vision.

T-cell response to structural and nonstructural hepatitis C virus antigens in persistent and self-limited hepatitis C virus infections.

Twenty-nine patients with chronic hepatitis C and 15 asymptomatic hepatitis C virus antibody-positive subjects who clinically recovered from hepatitis C virus infection were studied for their peripheral blood lymphomononuclear cell proliferative response to hepatitis C virus structural and nonstructural antigens (core, envelope, nonstructural 4 and nonstructural 5) expressed in yeast as superoxide dismutase fusion proteins, in an initial attempt to define some of the features of the virus-specific immune response. Hepatitis C virus core was the most immunogenic antigen for human leukocyte antigen class II-restricted T cells in both groups of patients studied, and the proliferative response to it was the most vigorous and the most frequently expressed in comparison with the other antigens tested. The specificity of the results was supported by the lack of response to hepatitis C virus antigens by healthy uninfected controls and confirmed by recognition of recombinant core proteins of different origin (yeast and baculovirus) by polyclonal T-cell lines produced by T-cell stimulation with yeast-derived core. Each of the antigens tested was able to induce significant although variable levels of proliferative response, indicating that all can be immunogenic at the T-cell level. Significant proliferative responses to core, nonstructural 4 and nonstructural 5 antigens were more frequently detected in subjects who were able to eradicate infection than in patients with chronic hepatitis C, although the difference was statistically not significant. No difference was observed between the two groups of patients with respect to the response to the putative envelope antigens.

Definition of a minimal optimal cytotoxic T-cell epitope within the hepatitis B virus nucleocapsid protein.

Residues 11 to 27 of the hepatitis B virus nucleocapsid antigen contain a cytotoxic T-cell epitope that is recognized by cytotoxic T cells from virtually all HLA-A2-positive patients with acute hepatitis B virus infection. Using panels of truncated and overlapping peptides, we now show that the optimal amino acid sequence recognized by cytotoxic T cells is a 10-mer (residues 18 to 27) containing the predicted peptide-binding motif for HLA-A2 and that this peptide can stimulate cytotoxic T cells able to recognize endogenously synthesized hepatitis B core antigen. Since patients with chronic hepatitis B virus infection fail to mount an efficient cytotoxic T-cell response to it, this epitope might serve as the starting point for the design of synthetic peptide-based immunotherapeutic strategies to terminate persistent viral infection.