Diagnostic Utility of Biplex/Multiplex Polymerase Chain Reaction in Infectious Granulomatous Dermatitis in North Indian Population.

BACKGROUND: A definite diagnosis of infectious granulomatous dermatitis (IGD) is difficult for both practicing dermatologists and dermatopathologists due to overlapping clinical and histomorphological features. We aimed to explore the role of multiplex polymerase chain reaction (PCR) for identifying a definite etiological agent for diagnosis and appropriate treatment in IGD in formalin-fixed paraffin-embedded tissue. MATERIALS AND METHODS: Sixty-two cases of IGD were included, excluding leprosy. The histochemical stains including Ziehl-Neelsen, periodic acid-Schiff, and Giemsa were performed in all cases. A multiplex PCR was designed for detection of tuberculosis (TB) (IS6110 and mpt64), fungal infections (ITS1, ITS2; ZM1, and ZM3), and leishmaniasis (kDNA). The results of histomorphology, histochemical stains, and multiplex PCR were compared. RESULTS: Among 62 cases, the sensitivity rate of PCR detection for organisms was 16.7%, 0%, 100%, 72%, 75%, and 66.7% in patients with TB, suggestive of TB, leishmaniasis, fungal infections, and granulomatous dermatitis not otherwise specified and granulomatous dermatitis suggestive of fungus, respectively. The TB PCR using IS6110 primers was negative in all cases; however, PCR using mpt64 primers was positive in 33.33% cases of scrofuloderma. The histochemical stains including Ziehl-Neelsen for acid-fast bacilli, periodic acid-Schiff for fungus, and Giemsa for Leishman-Donovan bodies showed positivity in 11.3%, 43.5%, and 3.2%, respectively. CONCLUSION: A multiplex PCR (Mycobacterium tuberculosis, Leishmania, and panfungal) is highly recommended in all cases of IGD where an etiological agent is difficult to establish by skin biopsy and histochemical stains along with a clinicopathological correlation. This will augment in appropriate treatment and will reduce empirical treatment and morbidity in such patients.

Diagnóstico diferencial de tuberculosis en base a PCR en lesiones granulomatosas clasificadas histopatológicamente / PCR-based differential diagnosis of tuberculosis in histopathologically classified granulomatous lesions

El diagnóstico diferencial de tuberculosis en tejidos fijados en formalina e incluidos en parafina es necesario porque la morfología de la lesión tuberculosa es variada, hay diversos granulomas clasificados en necrobióticos, tuberculoideos, supurativos, sarcoideo, a cuerpo extraño/crónico inespecífico. Las lesiones granulomatosas ocurren en tuberculosis y también en otras infecciones (hongos, parásitos, brucelosis, lepra) en condiciones tóxicas, alérgicas, autoinmunes, tumores y otras. El diagnóstico histológico no es confirmatorio de tuberculosis y en ausencia de una baciloscopia positiva, se hace necesaria la confirmación molecular para el diagnóstico diferencial. Evaluamos la eficacia de la técnica de PCR para la detección de tuberculosis en tejidos fijados y comparamos esos resultados con la histología del granuloma y la baciloscopia. Analizamos 444 biopsias de diferentes tejidos (ganglios, piel, pleura, pulmón, intestino, tejido óseo, mama y otros) de 5 tipos de granulomas: G1.tuberculoideo con necrosis caseosa; G2.tuberculoideo sin necrosis caseosa; G3. supurativo; G4. sarcoideo l; G5. a cuerpo extraño/inespecífico. Utilizamos dos PCR-IS6110 nested para detección del complejo Mycobacterium tuberculosis y un pan PCR-hsp65 nested para detección de Mycobacterium spp. Los resultados obtenidos muestran que la detección de tuberculosis mediante PCR fue significativamente superior que mediante baciloscopia. G1: PCR 69,6%, baciloscopia 31,3%; G2: PCR 26,8%, baciloscopia 6,1%; G3: PCR 16,7%, baciloscopia 6,7%; G4: PCR 7%, baciloscopia 4%; G5: PCR 6,7%, baciloscopia 0%. Concluimos que el diagnóstico molecular de tuberculosis mediante un PCR robusto adaptado a tejidos fijados es eficaz, rápido, sensible y contribuye a la precisión del diagnóstico diferencial en diferentes tipos de granulomas (AU)

The differential diagnosis of tuberculosis in fixed paraffin embedded-tissues is necessary due to both the diverse morphology of tuberculous lesions and the varying histological types of granulomas (necrobiotic, tuberculoid, suppurative, sarcoidal and foreign body/inespecific). Granulomatous lesions occur in tuberculosis, in other infections (fungal, parasitic, brucelosis, lepra), in toxic, allergic and autoimmune, tumours and in conditions of unknown etiology. Diagnosis of tuberculosis cannot be confirmed by histopathology alone and in absence of a positive acid-fast bacilli (AFB) stain, molecular confirmation of tuberculosis is necessary for a correct differential diagnosis. The aim of our study was to assess PCR efficacy for mycobacterial infection detection in fixed tissues and to correlate those findings with granuloma histology and with AFB staining. We analyzed 444 biopsies from various tissues (lymph nodes, skin, pleura, lung, intestine, bone tissue, breast and others) with 5 granuloma types: G1: with caseous necrosis; G2: without caseous necrosis; G3: suppurative; G4: sarcoidal; G5: chronic/nonspecific. For molecular detection, we used nested PCR-IS6110 for Mycobacterium tuberculosis complex and a nested pan PCR-hsp65 for Mycobacterium sp.. The results obtained demonstrated that PCR was significantly better than AFB stain for tuberculosis detection. G1: PCR 69.6%, AFB staining 31.3%. G2: PCR 26.8%, AFB staining 6.1%; G3: PCR 16.7%, AFB staining 6.7%; G4: PCR 7%, AFB staining 4%. G5: PCR 6.7%, AFB staining 0%. We conclude that molecular diagnosis of tuberculosis using robust PCR-based testing adapted to fixed tissues is a fast, efficient and sensitive method that increases the accuracy of the differential diagnosis of granulomatous lesions (AU)

Association of DRB1*1501 with disseminated Mycobacterium avium complex infection in North American AIDS patients.

The HLA class II allele, DR2 (DRB1*1501), has been repeatedly found to be associated with development of tuberculosis and leprosy. We searched for associations of these and other class II alleles with disseminated Mycobacterium avium complex infection (DMAC) in North American Caucasian homosexual AIDS patients. Molecular typing for HLA-DRB1 and -DQB1 alleles in 176 cases of DMAC and 176 matched controls showed an association of accelerated onset of disease with DRB1*1501 (and the closely linked DQB1*0602) that was stronger upon adjustment for the degree and duration of CD4+ cell deficiency (P=0.04) and in multivariate analysis (P=0.02) than in unadjusted analysis. A similar trend was seen with DRB1*0701, and no other allele showed a relationship of similar magnitude. M. avium complex organisms may more effectively evade host defenses in individuals carrying an HLA polymorphism identical to that associated with M. tuberculosis and M. leprae.

Immunogenetics of uveitis in leprosy.

PURPOSE: To identify any possible determinants in the development of uveitis in leprosy patients. METHODS: Human leukocyte antigen (HLA) class I and II antigen, and HLA class II genotypings were analyzed among Japanese leprosy patients. Ninety-three unrelated Japanese leprosy patients (46 patients with a history of uveitis and 47 patients without uveitis) and 114 healthy control subjects were investigated. RESULTS: The occurrence of HLA-DR2 was significantly higher in patients with uveitis (78.3%) than in those without uveitis (57.4%; odds ratio = 2.7, P<.05) and in the controls (33.3%; odds ratio = 7.2, P<.0000005, Pc<.00005). The occurrence of HLA-DR4 was significantly lower in patients with uveitis (15.2%) than in those without it (38.3%; odds ratio = 0.29, P<.05) and in the controls (46.5%; odds ratio = 0.21, P<.0005, Pc<.05). Furthermore, the frequencies of DR2-positive and DR4-negative genotypes were significantly higher in patients with uveitis (69.6%) than in those without it (38.3%; odds ratio = 3.7, P<.005) and in the controls (21.9%; odds ratio = 8.1, P<.00000005). At the genomic level, the occurrence of HLA-DQB1*0302 was significantly lower in the patients with uveitis (8.7%) than in those without it (25.5%; odds ratio = 0.28, P<.05). The distribution of HLA-DRB1 and DQA1 alleles was not significantly different between the patients with and those without uveitis. However, the frequencies of DRB1*1501-positive, as well as DRB1*0405- and DQB1*0302-negative genotypes were significantly higher in the patients with uveitis (47.8%) than in those without it (25.5%; odds ratio = 2.7, P<.05) and in the controls (8.8%; odds ratio = 9.5, P<.00000005). CONCLUSIONS: Our results suggest that HLA Class II genes confer susceptibility to or protection from leprous uveitis.

Immunogenetics of episcleritis in leprosy.

Human leukocyte antigens (HLA) were analyzed among Japanese leprosy patients to identify any possible determinants in the development of episcleritis in leprosy patients. Seventy-nine Japanese leprosy patients (33 patients with history of episcleritis and 46 patients without episcleritis) and 114 healthy control subjects were investigated. Human leukocyte antigen-class I and class II specificities were determined serologically by the standard microcytotoxicity test. The HLA-DRB1, -DRB5, -DQA1, and -DQB1 genotypings were performed by using the polymerase chain reaction (PCR)-single strand conformation polymorphism and PCR-restriction fragment length polymorphism analyses. The frequency of HLA-Cw3 was significantly increased among the patients with episcleritis (66.7%) compared to patients without episcleritis (43.5%; odds ratio = 2.6, P < 0.05). The frequency of HLA-DR4 was significantly decreased among the patients with episcleritis (15.2%) compared to patients without episcleritis (39.1%; odds ratio = 0.28, P < 0.05) and the controls (46.5%; odds ratio = 0.21, P < 0.001). At the genomic level, frequencies of the HLA-DRB1*0405, -DQB1*0401, and -DQB1*0302 alleles were significantly decreased among the patients with episcleritis (0%, 0%, and 6.1%, respectively) compared to patients without episcleritis (15.2%, 13.0%, and 26.1%, respectively; odds ratio = 0.07, 0.09, and 0.18, P < 0.05). HLA-DRB1*0405 and -DQB1*0401 were also significantly decreased among the patients with episcleritis compared to the controls (29.8% and 29.8%; odds ratio = 0.04, P < 0.0001). Our results suggest that HLA-Cw3 antigen confers the susceptibility to the development of episcleritis among Japanese leprosy patients. Concurrently, the DRB1 (the -DBR1*0405), and/or DQB1 (the -DQB1*0401 and -DQB1*0302) alleles might provide protection against leprous episcleritis.

HLA antigens in Omanis with blinding trachoma: markers for disease susceptibility and resistance.

AIM: To determine the presence of HLA antigens in people with blinding trachoma. METHODS: Fifty Omanis with blinding trachoma were serologically typed for HLA A, B, C, DR, and DQ antigens and DNA typed for class II DR beta and DQ beta alleles and compared with a population of 100 healthy controls. RESULTS: chi 2 analysis of serological reactions did not reveal any significant differences in HLA antigen frequencies after correction of probability, although DR4, DR7, and DR53 were completely absent in the patients and all of the patients were HLA DQ1 positive. In the case of DQ1 the relative risk was 22.6 (95% confidence interval of 20.7-24.7). Class II DNA low resolution DR beta typing showed a significant increase in HLA DR16 (pc = 0.036, relative risk = 3.8) and a significant decrease in HLA DR53 (pc = 0.018, relative risk = 0.05). CONCLUSION: The finding that HLA DR16 (a DR2 subtype) is associated with susceptibility to blinding trachoma, a disease that is caused by an intracellular micro-organism, is consistent with reports of an HLA DR2 association with leprosy and tuberculosis, diseases also caused by an intracellular micro-organism. Similarly, resistance to leprosy is associated with HLA DR53 as is the case with blinding trachoma described here. It is postulated that HLA DR2 or subtypes in association with HLA DQ 1 may enable an intracellular micro-organism to enter the cell or are involved in presentation of peptides derived from intracellular micro-organisms to T lymphocytes initiating a delayed hypersensitivity or autoimmune reaction. These findings are the first report that genetic factors are of major importance in the development and protection against blinding trachoma.

On the etiology of Crohn disease.

Crohn disease (CD) is a chronic, panenteric intestinal inflammatory disease. Its etiology is unknown. Analogous to the tuberculoid and lepromatous forms of leprosy, CD may have two clinical manifestations. One is aggressive and fistulizing (perforating), and the other is contained, indolent, and obstructive (nonperforating) [Gi]-berts, E. C. A. M., Greenstein, A. J., Katsel, P., Harpaz, N. & Greenstein, R. J. (1994) Proc. Natl. Acad. Sci. USA 91, 12721-127241. The etiology, if infections, may be due to Mycobacterium paratuberculosis. We employed reverse transcription PCR using M. paratuberculosis subspecies-specific primers (IS 900) on total RNA from 12 ileal mucosal specimens (CD, n = 8; controls, n = 4, 2 with ulcerative colitis and 2 with colonic cancer). As a negative control, we used Myobacterium avium DNA, originally cultured from the drinking water of a major city in the United States. cDNA sequence analysis shows that all eight cases of Crohn's disease and both samples from the patients with ulcerative colitis contained M. paratuberculosis RNA. Additionally, the M. avium control has the DNA sequence of M. paratuberculosis. We demonstrate the DNA sequence of M. paratuberculosis from mucosal specimens from humans with CD. The potable water supply may be a reservoir of infection. Although M. paratuberculosis signal in CD has been previously reported, a cause and effect relationship has not been established. In part, this is due to conflicting data from studies with empirical antimycobacterial therapy. We conclude that clinical trials with anti-M. paratuberculosis therapy are indicated in patients with CD who have been stratified into the aggressive (perforating) and contained (nonperforating) forms.

Mosaic evolution of prepropancreatic polypeptide.

Pancreatic polypeptide, a 36-amino acid peptide hormone, is synthesized in pancreatic islets of Langerhans and acts as a regulator of pancreatic and gastrointestinal functions. We isolated cDNA clones encoding rat pancreatic polypeptide precursor from an islet cDNA library and determined their nucleic acid sequences. Rat pancreatic polypeptide was found to be flanked on the amino terminus by a putative signal peptide and on the carboxyl terminus by Gly-Lys-Arg followed by a 30-amino acid peptide. Nucleotide and amino acid sequences of the signal peptide and the pancreatic polypeptide of the rat were highly homologous to those of the human (Boel, E., Schwartz, T. W., Norris, K. E., and Fill, N. P. (1984) EMBO J. 3, 909-912). On the other hand, the rat carboxyl-terminal peptide differed markedly from the corresponding domain of the human precursor and did not contain any sequence similar to the icosapeptide, which has so far been known to be a second stable product from mammalian pancreatic polypeptide precursors (Schwartz, T. W., Hansen, H. F., Hakanson, R., Sundler, F., and Tager, H. S. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 708-712). The mosaicism of sequence conservation and divergence in prepropancreatic polypeptides may be a unique example in the evolution of prohormones.

Leprosy in a mangabey monkey--naturally acquired infection.

Naturally acquired leprosy was detected in an otherwise normal "sooty" mangabey monkey (Cercocebus atys). This animal was imported from West Africa in 1975 and developed clinical symptoms of leprosy in 1979. Histopathologic findings were those of subpolar-lepromatous to borderline-lepromatous leprosy in the Ridley-Jopling classification. The disease was progressive, with crippling neuropathic deformities of the hands and feet. The disease regressed under specific therapy. The etiologic agent was identified as Mycobacterium leprae by the following criteria: invasion of nerves of host, staining properties, electron microscopic findings, noncultivable on mycobacteriologic media, DOPA-oxidase positive, lepromin reactivity, infection patterns in mice and armadillos, sensitivity to sulfone, and DNA homology. We believe the animal acquired the disease from a patient with active leprosy. The mangabey monkey offers promise as a primate model for leprosy, and adds a third reported species to animals with naturally acquired leprosy.

The placenta in leprosy.

Eighty-one placentae from women with leprosy and 17 placentae from healthy controls were subjected to a detailed macroscopic, light microscopic, ultrastructural, immunopathological, microbiological and biochemical study. The placental morphology and immunohistology were normal, and there was no morphological evidence of infection of the placenta due to M. leprae. No acid-fast bacilli or acid-fast bacillary granules were seen on light microscopy of any of the placentae from leprous women, although homogenates from two out of seven placentae from women with very active lepromatous leprosy contained acid-fast bacilli in very small numbers. The small placental size of women with leprosy, most marked in those with lepromatous leprosy, appears to be due to a decrease in placental cell size, rather than to a reduced number of cells in the placenta. It is postulated that the small placenta and reduced fetal birth weight observed in lepromatous leprosy are a consequence of depressed maternal immune reactivity.

Connective tissue aging in the human hypophysis-gonadal system.

The basic issue in our project was to quantify the age-process in the human hypophysis-gonadal system. Of the many aspects of aging, we estimated, using human autopsy material, the content of desoxyribonucleic acid (DNA), of hydroxyproline, of hexosamines and of uronic acids in the hypophysis, testis, and ovary. The respective organs were weighed and homogenised in acid-buffer solution after which, for each of the parameters, 1.0 ml of homogenate was extracted and processed: DNA-estimation according to modified Dische/Seibert (1929) method; hydroxy-proline according to Stegemann and Stalder (1967); hexosamines and uronic acids according to Gatt and Berman (1966) and Blumenkrantz and Asboe-Hansen (1973) respectively. All values obtained were expressed in micrograms/ml of homogenate. The age investigated ranged from a few days old to 99 years. The DNA-curves for the hypophysis initially showed a decline till about the third decade of life and then remained almost constant with a significant decline up to the senile age. The DNA-curves for the testis and ovary demonstrated a sharp decline initially till the third decade of life, after which the curves remained constant, but showed reduction during higher age. Similar pattern of distribution was also observed for hexosamines, and uronic acids: after the initial decline of uronic acids in the human hypophysis and testis during the first three decades of life and nearly constant values thereafter, hexosamines, together with the uronic acids show minimal decrease in content during the senile period. Contents od hydroxyproline significantly increased in testis, ovary and hypophysis with aging. This biochemically proven age-fibrosis in the human gonads corresponds to the morphologically observed vascular and interstitial sclerosis in the testis and the ovary and may partly help to explain the declined sensitivity of these target organs to hypophyseal stimulation during the aging period.

Systematics of Hanseniaspora Zikes and Kloeckera Janke.

The physiological and morphological characteristics of eighty-two strains of Hanseniaspora and Kloeckera, represented twenty-nine described species, were examined. These results along with DNA base composition and DNA/DNA reassociation experiments revealed that the genus Hanseniaspora comprises six distinct species, viz. H. valbyensis, H. uvarum, H. guilliermondii, H. occidentalis, H. osmophila and H. vineae, with K. japonica, K. apiculata, K. apis, K. javanica, K. corticis and K. africana, respectively, as their imperfect states.

Kloeckera apis st. nov.; the imperfect state of Hanseniaspora guilliermondii Pijper.

K. apiculata var. apis (nom. nud.) was found to be the imperfect state of H. guilliermondii Pijper by a high degree of DNA reassociation. The name is validated and raised to species rank, K. apis Lavie ex Smith, Simione and Meyer. K. apis and H. guilliermondii could be distinguished from H. uvarum and H. valbyensis by a low DNA reassociation and by growth at 37 C.

Clofazimine binding studies with deoxyribonucleic acid.

1. The antileprosy drug, clofazimine, formed stable complexes with DNA and transfer RNA. A quantitative study was made of the spectral red shifts that occurred when clofazimine interacted with DNA. The red shift appeared specific for clofazimine binding to nucleic acid polymers. 2. The degree of clofazimine interaction with DNA was related to the G+C content of the DNA strand. As compared to the human strand, clofazimine interacted with the mycobacterial strand to give a larger red shift which was consistent with the increased G+C content of mycobacterial DNA. 3. It was found that clofazimine interacted with the synthetic single-stranded polynucleotide, poly G, whereas little interaction occurred withpoly A, poly C, or poly U. It was concluded that the guanine base region was a predominant site of clofazimine binding to DNA. 4. No evidence was found to indicate that clofazimine underwent intercalative binding between the base pairs of DNA. 5. It was proposed that clofazimine underwent binding along the minor groove region of DNA at appropriate base sequences which contain guanine. The resultant effect would inhibit template function of the DNA strand.