Quantitative polymerase chain reaction in paucibacillary leprosy diagnosis: A follow-up study.

OBJECTIVE: The diagnosis of paucibacillary (PB) leprosy cases remains a challenge because of the absence of a confirmatory laboratory method. While quantitative polymerase chain reaction (qPCR) has been shown to provide reliable sensitivity and specificity in PB diagnoses, a thorough investigation of its efficacy in clinical practice has not yet been published. The present study evaluated patients with suspected leprosy skin lesions by using qPCR to identify PB individuals in the Leprosy Outpatient clinic at the Oswaldo Cruz Foundation in Rio de Janeiro, Brazil. METHODS: One hundred seventy-two suspected PB cases were included in the study. The patients were evaluated by a dermatologist at three different times. The clinical dermato-neurological examination and collected samples were performed on the first visit. On the second visit, the results of the histopathological analysis and PCR assay (DNA-based Mycobacterium leprae qPCR-targeting 16S gene) results were analyzed, and a decision regarding multi-drug therapy was made. A year later, the patients were re-examined, and the consensus diagnosis was established. RESULTS: In 58% (100/172) of cases, a conclusive diagnosis via histopathological analysis was not possible; however, 30% (30/100) of these cases had a positive PCR. One hundred ten patients (110/172) attended the third visit. The analysis showed that while the sensitivity of the histopathological test was very low (35%), a qPCR alone was more effective for identifying leprosy, with 57% sensitivity. CONCLUSION: The use of qPCR in suspected PB cases with an inconclusive histology improved the sensitivity of leprosy diagnoses.

Toward the identification, characterization and experimental culture of Lacazia loboi from Atlantic bottlenose dolphin (Tursiops truncatus).

Lobomycosis (lacaziosis) is a chronic, granulomatous, fungal infection of the skin and subcutaneous tissues of humans and dolphins. To date, the causative agent, the yeast-like organism Lacazia loboi, has not been grown in the laboratory, and there have been no recent reports describing attempts to culture the organism. As a result, studies on the efficacy of therapeutics and potential environmental reservoirs have not been conducted. Therefore, the objective of the current study was to utilize both classical and novel microbiological methods in order to stimulate growth of Lacazia cells collected from dolphin lesions. This included the experimental inoculation of novel media, cell culture, and the use of artificial skin matrices. Although unsuccessful, the methods and results of this study provide important insight into new approaches that could be utilized in future investigations of this elusive organism.

Detection of Multiple Budding Yeast Cells and a Partial Sequence of 43-kDa Glycoprotein Coding Gene of Paracoccidioides brasiliensis from a Case of Lacaziosis in a Female Pacific White-Sided Dolphin (Lagenorhynchus obliquidens).

Lacaziosis, formerly called as lobomycosis, is a zoonotic mycosis, caused by Lacazia loboi, found in humans and dolphins, and is endemic in the countries on the Atlantic Ocean, Indian Ocean and Pacific Ocean of Japanese coast. Susceptible Cetacean species include the bottlenose dolphin (Tursiops truncatus), the Indian Ocean bottlenose dolphin (T. aduncus), and the estuarine dolphin (Sotalia guianensis); however, no cases have been recorded in other Cetacean species. We diagnosed a case of Lacaziosis in a Pacific white-sided dolphin (Lagenorhynchus obliquidens) nursing in an aquarium in Japan. The dolphin was a female estimated to be more than 14 years old at the end of June 2015 and was captured in a coast of Japan Sea in 2001. Multiple, lobose, and solid granulomatous lesions with or without ulcers appeared on her jaw, back, flipper and fluke skin, in July 2014. The granulomatous skin lesions from the present case were similar to those of our previous cases. Multiple budding and chains of round yeast cells were detected in the biopsied samples. The partial sequence of 43-kDa glycoprotein coding gene confirmed by a nested PCR and sequencing, which revealed a different genotype from both Amazonian and Japanese lacaziosis in bottlenose dolphins, and was 99 % identical to those derived from Paracoccidioides brasiliensis; a sister fungal species to L. loboi. This is the first case of lacaziosis in Pacific white-sided dolphin.

Leprous neuromyositis: A rare clinical entity and review of the literature.

Mycobacterium leprae, the causative agent of leprosy (Hansen's disease), is a slow growing intracellular acid-fast bacillus that affects the skin, peripheral nerves and respiratory tract. In patients with suppressed cell-mediated immunity, the infiltration of the Bacilli can produce disseminated illness such as leprous neuromyositis. We reported a case of 56-year-old gentleman presenting with pyrexia of unknown origin, asymmetric sensory motor axonal polyneuropathy and was on chronic exogenous steroid therapy. On evaluation, his skin, muscle, nerve and bone marrow biopsy showed numerous globi of acid-fast Bacilli suggestive of leprous neuromyositis, a rare form of disseminated Hansen's disease. We reported this case in view of its rarity, atypical manifestation of a relatively rare disease and literature review on poor electrophysiological correlation in the diagnosis of leprous neuromyositis as compared to the histopathological examination.

Study of murine experimental Jorge Lobo's disease by analysis of peritoneal lavage cells and footpad histopathology: early versus chronic lesions.

The murine model of Jorge Lobo's disease is characterized by histological alterations similar to those seen in human disease, including a large number of viable fungi. This study evaluated the immune response of mice with early and late macroscopic lesions (5 and 13 months post-inoculation [p.i.], respectively) by the analysis of peritoneal lavage cells and footpad (FP) histology. The FP of mice were inoculated with 1 × 10(6) fungi (viability index of 41%). At 5 and 13 months p.i., the granuloma mainly consisted of macrophages and multinucleated giant cells, but a larger number of neutrophils was observed at 5 months and lymphocytes at 13 months. The number of fungi in the FP and fungal viability were 1.8 ± 1.1 × 10(6) fungi/ml and 38.5% at 5 months p.i. and 30.8 ± 11.7 × 10(6) fungi/ml and 9% at 13 months (P < .05). Higher production of H2O2, O2(-), IL-10, and TNF-α were observed at 13 months (P < .05), but there was no significant difference in the production of NO, IL-2, IL-4, IL-12 and IFN-γ. The results showed significant differences between early and late lesions and support the use of BALB/c mice for evaluation of the different phases of infection.

Study of murine experimental Jorge Lobo's disease by analysis of peritoneal lavage cells and footpad histopathology: early versus chronic lesions

The murine model of Jorge Lobo's disease is characterized by histological alterations similar to those seen in human disease, including a large number of viable fungi. This study evaluated the immune response of mice with early and late macroscopic lesions (5 and 13 months post-inoculation [p.i.], respectively) by the analysis of peritoneal lavage cells and footpad (FP) histology. The FP of mice were inoculated with 1 × 106 fungi (viability index of 41%). At 5 and 13 months p.i., the granuloma mainly consisted of macrophages and multinucleated giant cells, but a larger number of neutrophils was observed at 5 months and lymphocytes at 13 months. The number of fungi in the FP and fungal viability were 1.8 ± 1.1 × 106 fungi/ml and 38.5% at 5 months p.i. and 30.8 ± 11.7 × 106 fungi/ml and 9% at 13 months (P < .05). Higher production of H2O2, O2−, IL-10, and TNF-α were observed at 13 months (P < .05), but there was no significant difference in the production of NO, IL-2, IL-4, IL-12 and IFN-γ. The results showed significant differences between early and late lesions and support the use of BALB/c mice for evaluation of the different phases of infection

Mycobacterium leprae and Mycobacterium haemophilum co-infection in an iatrogenically immunosuppressed patient.

We present the case of a native Texan who was diagnosed with tuberculoid leprosy and later developed a cutaneous infection with M. haemophilum following iatrogenic immunosuppression. To our knowledge, there are no such reports of M. haemophilum and M. leprae infection occurring simultaneously in the same host.

Multiple nerve abscesses on cutaneous radial nerve--a case of pure neural leprosy.

Leprosy is a unique infectious disease due to varied spectrum of clinical signs it exhibits. Pure neural leprosy (PNL) is an unusual form of leprosy and accounts for 4-8% of all leprosy cases. It can manifest as a simple tingling sensation to complex and tragic motor paralysis. Here we report a case of PNL involving isolated cutaneous radial nerve as multiple abscesses along the course of the nerve. To the best of our knowledge, this is rarest presentation of pure neural leprosy.

Late onset neuropathy in leprosy patients released from treatment: not all due to reactions?

OBJECTIVES: To evaluate the clinical, neurophysiological and histological features of cases of neuropathy developing after completion of anti-leprosy treatment, where biopsy showed inflammatory changes. PATIENTS AND METHODS: Seven patients were evaluated by a single neurologist. Electro-neuro-myography and peripheral nerve biopsy were performed in all patients. RESULTS: Median age was 50-6 years. Time from release from treatment and onset of symptoms ranged from 1 to 12 years (median of 6.6 years). Sensory symptoms were the most common complaint, including pain (71%) and paresthesiae (71%). Muscle weakness was found in 51% and muscle atrophy in 43% of the subjects. Peripheral nerve thickening was present in all patients. Neurophysiological studies suggested sensory-motor polyneuropathy and multiple mono-neuropathy. Nerve biopsy showed inflammatory processes with fibrosis of endoneurium, perineurium and epineurium and total or partial loss of fibres. No bacilli were detected with Wade staining. Patients treated with corticosteroids had some relief of symptoms. CONCLUSION: After release from treatment, leprosy patients may insidiously develop progressive peripheral nerve symptoms not fulfilling criteria for relapse or leprosy reactions. Sensory symptoms predominate and peripheral nerve thickening is an important finding. We speculate that these late onset symptoms are secondary to chronic immune-mediated processes in response to antigens of M. leprae.

Ackerman's tumour of buccal mucosa in a leprosy patient.

Leprosy (Hansen's disease) is a chronic granulomatous disease caused by Mycobacterium leprae (Hansen's bacillus). Oral manifestations occur in 20-60% of cases, usually in lepromatous leprosy, and are well documented. They may involve both the oral hard and soft tissues. Incidence of verrucous carcinoma/Ackerman's tumour developing in anogenital region and plantar surfaces of feet in lepromatous leprosy has been sufficiently documented in the literature. However, association of oral verrucous carcinoma with lepromatous leprosy has not been established. We report for the first time a case of verrucous carcinoma of the buccal mucosa occurring in a leprotic patient, with brief review of literature on orofacial manifestations of leprosy.

Type I reaction in leprosy--a histopathological analysis.

Even though type 1 lepra reaction (TIR) is a commonly encountered clinical problem, its histology has not yet been clearly delineated. This study attempts to enumerate the most sensitive parameters for the histological diagnosis of TIR. Case records between March 2007 and September 2007 of patients with TIR were reviewed and the biopsies were evaluated by a pathologist blinded to the previous diagnoses. Twenty three patients were included in the study. The most sensitive parameters in our study were dermal edema, intra-granuloma edema and giant cell size. Though clinical findings should remain the mainstay of diagnosis of TIR, the above mentioned parameters should be evaluated in biopsies of leprosy to look for signs of reaction which might otherwise be missed.

Lepromatous leprosy with an uncommon presentation: a case report.

28 yr old male presented with asymptomatic nodules and few well to ill defined papules on ears, asymmetrical nerve enlargement and evanescent tender nodules on the extremities without any infiltration of the skin and madarosis. Slit skin smear done from normal skin was BI 6+. Skin biopsy showed features of lepromatous leprosy.

Microbiological, histological, immunological, and toxin response to antibiotic treatment in the mouse model of Mycobacterium ulcerans disease.

Mycobacterium ulcerans infection causes a neglected tropical disease known as Buruli ulcer that is now found in poor rural areas of West Africa in numbers that sometimes exceed those reported for another significant mycobacterial disease, leprosy, caused by M. leprae. Unique among mycobacterial diseases, M. ulcerans produces a plasmid-encoded toxin called mycolactone (ML), which is the principal virulence factor and destroys fat cells in subcutaneous tissue. Disease is typically first manifested by the appearance of a nodule that eventually ulcerates and the lesions may continue to spread over limbs or occasionally the trunk. The current standard treatment is 8 weeks of daily rifampin and injections of streptomycin (RS). The treatment kills bacilli and wounds gradually heal. Whether RS treatment actually stops mycolactone production before killing bacilli has been suggested by histopathological analyses of patient lesions. Using a mouse footpad model of M. ulcerans infection where the time of infection and development of lesions can be followed in a controlled manner before and after antibiotic treatment, we have evaluated the progress of infection by assessing bacterial numbers, mycolactone production, the immune response, and lesion histopathology at regular intervals after infection and after antibiotic therapy. We found that RS treatment rapidly reduced gross lesions, bacterial numbers, and ML production as assessed by cytotoxicity assays and mass spectrometric analysis. Histopathological analysis revealed that RS treatment maintained the association of the bacilli with (or within) host cells where they were destroyed whereas lack of treatment resulted in extracellular infection, destruction of host cells, and ultimately lesion ulceration. We propose that RS treatment promotes healing in the host by blocking mycolactone production, which favors the survival of host cells, and by killing M. ulcerans bacilli.

Limited susceptibility of cynomolgus monkeys (Macaca fascicularis) to leprosy after experimental administration of Mycobacterium leprae.

Cynomolgus monkeys are a useful model for human tuberculosis, but susceptibility to M. leprae is unknown. A cynomolgus model of leprosy could increase understanding of pathogenesis-importantly, neuritis and nerve-damaging reactions. We administered viable Mycobacterium leprae to 24 cynomolgus monkeys by three routes, with a median follow-up period of 6 years (range = 1-19 years) involving biopsies, nasal smears, antiphenolic glycolipid-1 (PGL-1) antibody serology, and lepromin skin testing. Most developed evanescent papules at intradermal M. leprae inoculation sites that, on biopsy, showed a robust cellular immune response akin to a lepromin skin test reaction; many produced PGL-1 antibodies. At necropsy, four monkeys, without cutaneous or gross neurological signs of leprosy but with elevated PGL-1 antibodies, including three with nasal smears (+) for acid fast bacilli (AFB), showed histological features, including AFB, suggestive of leprosy at several sites. Overall, however, cynomolgus monkeys seem minimally susceptible to leprosy after experimental M. leprae administration.

Clinical presentation of tuberculoid leprosy in an epidermodysplasia verruciformis patient.

Epidermodysplasia verruciformis (EV) is triggered by a variety of mechanisms that at least partly include genetic background. We present a Brazilian man with a 30-year history of flat, wart-like lesions with clinical, histopathological, and evolutive aspects consistent with papillomavirus (HPV)-associated EV. Histological analysis of the wart lesions showed epidermis with hyperkeratosis, regular acanthosis, hypergranulosis, and cells with abundant basophilic cytoplasm. Moreover, a perivascular lymphocytic infiltrate was found in the superficial dermis, consistent with a viral wart. Type-2-HPV DNA was detected in various fragments of skin-wart lesions using the polymerase chain reaction (PCR). Two years after the EV diagnosis, the patient presented with an anesthetic well-demarcated, erythematous and mildly scaly plaque on his right forearm. A histopathological analysis of this lesion demonstrated the presence of a compact tuberculoid granuloma. Ziehl-Neelsen staining demonstrated the presence of rare acid-fast bacilli and confirmed the tuberculoid leprosy diagnosis. The patient's Mitsuda Intradermal Reaction was positive. To elucidate the possible mechanism involved in this case of EV, we genotyped the HLA genes of this patient. DQB genotyping showed the polymorphic HLA alleles DQB1*0301 and 0501. The patient was treated with a paucibacillary multi-drug therapy scheme, and the disease was cured in six months. This report describes an EV patient with an M. leprae infection, confirming that tuberculoid leprosy patients possess a relatively specific and efficient cell-mediated immunity against the bacillus and, therefore, localized forms of the disease. Moreover, we show the possible involvement of the polymorphic HLA alleles DQB1*0301 and 0501 in EV induction mechanisms.

The use of the microplate alamar blue assay (MABA) to assess the susceptibility of Mycobacterium lepraemurium to anti-leprosy and other drugs.

Although murine leprosy is no longer a common illness, our understanding of the biology of this disease is incomplete. One particular example of this concerns the etiologic agent Mycobacterium lepraemurium (MLM). MLM is a fastidious microorganism that is difficult to grow in axenic media; in a way, this has hampered attempts to thoroughly study its physiological and metabolic characteristics. MLM is an obligate intracellular bacillus that invades macrophages and replicates profusely with a generation time that oscillates between 0.5 and 11 days. In the present study, we have successfully maintained MLM alive for more than 12 days in vitro, providing us with an opportunity to study its susceptibility to several anti-leprosy agents and other drugs. To achieve this, we used a fluorescence reduction assay of alamar blue (a resazurin) in a microplate format (microplate-alamar-blue-assay; MABA), which is a highly sensitive, practical, and inexpensive method for assaying cell viability. We found that MLM was highly susceptible to clofazimine and rifampicin and was less susceptible to streptomycin, thiacetazone, kanamycin, dapsone, and ethionamide, in that order. MLM was not susceptible to four plant triterpenoids (oleanolic acid, neolignan-c, sitosterol, and ursolic acid) for which bactericidal activity has been reported in M. tuberculosis. Because the MABA has high sensitivity, it can be used to monitor the activity of microorganisms that are difficult to cultivate (such as M. lepraemurium), in response to various drugs, thus offering a method to complement the study of murine leprosy, about which many questions remain unanswered.