Rapid diagnosis of genitourinary tuberculosis by polymerase chain reaction and non-radioactive DNA hybridization.

OBJECTIVE: To establish a polymerase chain reaction (PCR) assay for the rapid detection and identification of mycobacteria in urine, and to assess the value of such assay in routine laboratory diagnosis of genitourinary tuberculosis. MATERIALS AND METHODS: Urine specimens from 1000 patients with clinical suspicion of urinary tuberculosis were examined. Two assays for the detection and identification of Mycobacterium tuberculosis (M. tuberculosis) complex and mycobacteria other than tuberculosis (MOTT) by non-radioactive DNA hybridization of PCR-product were applied. The first assay used PCR primers and probe derived from M. tuberculosis species-specific DNA insertion sequence, IS6110. The second utilized mycobacterium genus-specific sequence encoding ribosomal ribonucleic acid (16S rRNA). The results obtained by PCR were compared with those obtained by standard microbiological methods, acid-fast bacilli (AFB) stain and culture. RESULTS: Compared with cultures, the sensitivity of AFB staining was 52.07% and the specificity was 96.7%. In comparison to the results of culture, the overall sensitivity and specificity of the IS6110-PCR assay was 95.59% and 98.12% respectively. While the corresponding results for the 16S rRNA gene-PCR were 87.05% and 98. 9%. CONCLUSION: The high sensitivity and specificity in addition to the potential for rapid detection of mycobacteria, makes this test a useful tool in the clinical management of mycobacterial infection in urine. Urine specimens may contain M. tuberculosis and/or other mycobacteria; therefore, there are advantages in using genus-specific primers in parallel with species-specific primers in PCR assay.

Porphyrin metabolism in rats intaking chemical carcinogens.

This study was performed on rats receiving nitrosamine precursors as potent liver carcinogens in order to investigate and follow up the porphyrin metabolism during the intake of hepatocarcinogens. As clarified from our results, progressive increases in free erythrocyte porphyrins, erythrocyte protoporphyrins, haem content and hepatic total porphyrins were observed after 2 months of intaking these carcinogens and further increases were gradually observed in parallel with the continued intake of these chemicals, which was extended to 7 months. At the same time, such elevations were also observed in the activity of hepatic delta-aminolevulinic acid (ALA)-synthetase and uroporphyrinogen-1-synthetase either in hepatic tissues or erythrocytes of these carcinogen-subjected rats. However, significant inhibition was found in the activity of erythrocyte ALA-dehydratase, which reached 40.5% of the control values after 7 months. Therefore, these observations demonstrated that the intake of hepatocarcinogens may influence the rate of hepatic porphyrin and haem biosynthesis.

Selective in vivo tumor localization of uroporphyrin isomer I in mouse mammary carcinoma: superiority over other porphyrins in a comparative study.

Twenty-eight porphyrins were evaluated for tumor localization as delineated by fluorescence using a transplantable KHJJ mammary carcinoma in BALB/c mice as the tumor model. Five of the 28 porphyrins were found to localize and of these, one, i.e., uroporphyrin I (UROP I), showed a higher tumor:skin ratio than any of the others; moreover, as no measurable UROP I was present in the gut, the tumor:intestinal porphyrin ratio under the conditions of assay was infinity. Because hematoporphyrin derivative (HPD), a complex mixture of porphyrins has been studied extensively as a tumor localizer, we compared HPD with UROP I at differing doses (2-40 mg/kg) and at different times (3-96 h) following i.v. administration. Dose response curves showed tissue levels of porphyrin to plateau out at doses above 20 mg/kg. Peak tumor HPD and UROP I levels attained 6-18 h after i.v. administration (40 mg porphyrin/kg) were comparable, but tumor retention of HPD over the ensuing 96 h was higher. The ratio of UROP I in tumor compared to skin was significantly greater throughout the period of observation. At all times, no UROP I was detectable in gastrointestinal mucosa. At differing doses (10-40 mg/kg), the tumor:skin ratio for HPD ranged from 1.47-1.85, and for UROP I from 6.06-12.33. As a function of time (6-72 h), the tumor:skin ratios respectively were 1.03-2.38, and 11.9 to infinity. At all times, the tumor:colon mucosa ratio at different doses for HPD approached 1 and for UROP I was infinity. We conclude that the greater specificity of tumor uptake by UROP I and its lack of retention by gut mucosa warrants further study to determine its potential clinical application as a diagnostic marker, particularly for early mucosal cancer, and in photoradiation therapy.

The interaction of tumour-localizing porphyrins with collagen, elastin, gelatin, fibrin and fibrinogen.

We have already reported in Balb C mouse transplantable mammary carcinoma, that uroporphyrin I and III are superior as tumour localizers when compared to hematoporphyrin derivative and a derivative thereof, photofrin II. This study compares the binding of porphyrins to proteins which may be found in tumour cells or stroma to investigate whether there is a common binding determinant. Coproporphyrin III and deuteroporphyrin IX which are non-tumour localizing porphyrins, were also part of the comparative study. The interaction of these porphyrins with acid soluble collagen and acid insoluble collagen, elastin, and fibrin was evaluated, and the binding of uroporphyrin isomers I and III and deuteroporphyrin IX to gelatin and fibrinogen, was also determined. The results suggest that collagen, especially the acid soluble form, and gelatin preferentially bind the four porphyrins which localize in mammary carcinoma tissue. The well reported observations that malignant epithelial cells, including breast cancer, produce collagen and contain a rate-limiting enzyme in collagen biosynthesis would support the notion that de novo synthesis of this protein may in part govern the tumour uptake and retention of porphyrins. Elastin, fibrinogen and fibrin showed non-discriminant binding to the porphyrins under study.

Tumour localization of uroporphyrin isomers I and III and their correlation to albumin and serum protein binding.

We were the first to report the superiority of uroporphyrin I (UROP I) as a tumour localizer when compared to haematoporphyrin derivative (HPD). In this study, we compared both isomers of UROP, i.e. I and III, in a KHJJ mammary carcinoma mouse model. Six and 18 h after UROP administration, the tumour, skin and gut porphyrin (P) content was quantitated. Tumour UROP I levels were always at least 50% higher than UROP III in tumour, whereas both isomers were barely detectable in the skin and gastrointestinal tract. We then explored the possibility that tumour P uptake might relate in part to the affinity of circulating P to mouse serum proteins (MSP), in particular, the major binding protein constituent, albumin. Copro-P III, deutero-P 2,4 disulphonic acid (DP), proto-P IX (PP) and heptacarboxylic P I (Hepta I) which in our mouse tumour model do not localize in malignant tissue, were compared to UROP I and III. The P was mixed with 0.775 microM human serum albumin (HSA) at different molar ratios (HSA:P range 2-8) and the unbound P concentration quantitated using an Amicon CF-25 membrane cone with centrifugation. The percentage free P was significantly higher for UROP I (92-98%) than III (82-95%) and significantly more than that observed with non-tumour localizing P studied. Similar data were obtained with MSP. This is consistent with the notion that enhanced uptake and retention (particularly UROP I) by malignant neoplastic tissue might reflect a higher affinity for UROP by tumour constituents than by circulating proteins.(ABSTRACT TRUNCATED AT 250 WORDS)