William R. Faber

Comparison between Quantitative Nucleic Acid Sequence-Based Amplification, Real-Time Reverse Transcriptase PCR, and Real-Time PCR for Quantification of Leishmania Parasites

ABSTRACT DNA or RNA amplification methods for detection of Leishmania parasites have advantages regarding sensitivity and potential quantitative characteristics in comparison with conventional diagnostic methods but are often still not routinely applied. However, the use and application of molecular assays are increasing, but comparative studies on the performance of these different assays are lacking. The aim of this study was to compare three molecular assays for detection and quantification of Leishmania parasites in serial dilutions of parasites and in skin biopsies collected from cutaneous leishmaniasis (CL) patients in Manaus, Brazil. A serial dilution of promastigotes spiked in blood was tested in triplicate in three different runs by quantitative nucleic acid sequence-based amplification (QT-NASBA), quantitative real-time reverse transcriptase PCR (qRT-PCR), and quantitative real-time PCR (qPCR). In addition, the costs, durations, and numbers of handling steps were compared, and 84 skin biopsies from patients with suspected CL were tested. Both QT-NASBA and qRT-PCR had a detection limit of 100 parasites/ml of blood, while qPCR detected 1,000 parasites/ml. QT-NASBA had the lowest range of intra-assay variation (coefficients of variation [CV], 0.5% to 3.3%), while qPCR had the lowest range of interassay variation (CV, 0.4% to 5.3%). Furthermore, qRT-PCR had higher r2 values and amplification efficiencies than qPCR, and qPCR and qRT-PCR had faster procedures than QT-NASBA. All assays performed equally well with patient samples, with significant correlations between parasite counts. Overall, qRT-PCR is preferred over QT-NASBA and qPCR as the most optimal diagnostic assay for quantification of Leishmania parasites, since it was highly sensitive and reproducible and the procedure was relatively fast.

Polymorphism N248S in the Human Toll‐Like Receptor 1 Gene Is Related to Leprosy and Leprosy Reactions

We investigated the association between a polymorphism of a key innate immunity receptor, Toll-like receptor 1 (TLR1) N248S, and susceptibility to leprosy and its clinical presentation. TLR1 N248S has been shown elsewhere to diminish TLR1 signaling and subsequent leprosy disease. The homozygous genotype SS was more frequent (P=.012) and the heterozygous SN genotype was less frequent (P=.015) in patients with leprosy than in control subjects. Additional observed differences in allelic frequency in patients who experienced reversal reactions and/or erythema nodosum leprosum reactions indicates that altered TLR1 function, or at least a TLR1 N248S-linked trait, may affect the progression from infection to disease as well as the disease course and the risk of debilitating reactional episodes in this population.

Thalidomide Therapy:An Open Trial

ABSTRACT: In an open trial, 35 patients suffering from a variety of dermatologic conditions were treated with thalidomide. The drug was shown to be a valuable addition to the therapeutic armamentarium; however, the occurrence of a peripheral neuropathy will limit its use just as its teratogenic side effects have done for many years.

Immunological Crossreactivity of the Mycobacterium leprae CFP‐10 with its Homologue in Mycobacterium tuberculosis

Mycobacterium tuberculosis culture filtrate protein‐10 (CFP‐10) (Rv3874) is considered a promising antigen for the immunodiagnosis of tuberculosis (TB) together with early secreted antigens of M. tuberculosis (ESAT‐6). Both ESAT‐6 and CFP‐10 are encoded by the RD1 region that is deleted from all tested M. bovis bacille Calmette–Guérin (BCG) strains but present in M. leprae, M. tuberculosis, M. bovis, M. kansasii, M. africanum and M. marinum. In this study, the homologue of CFP‐10 in M. leprae (ML0050) is identified and characterized. Interferon‐γ production in response to this homologue by T cells from leprosy patients, TB patients and unexposed controls shows that CFP‐10 of M. leprae is a potent antigen that crossreacts with CFP‐10 of M. tuberculosis at the T‐cell level. This crossreactivity has implications for the use of CFP‐10 of these mycobacterial species as diagnostic tool in areas endemic for both the diseases.

Imported Tropical Infectious Ulcers in Travelers

Skin ulcers are a commonly encountered problem at departments of tropical dermatology in the Western world. Furthermore, the general dermatologist is likely to be consulted more often for imported chronic skin ulcers because of the ever-increasing travel to and from tropical countries. The most common cause of chronic ulceration throughout the world is probably pyoderma. However, in some parts of the world, cutaneous leishmaniasis is one of the most prevalent causes. Mycobacterium ulcerans is an important cause of chronic ulcers in West Africa.Bacterial infections include pyoderma, mycobacterial infections, diphtheria, and anthrax. Pyoderma is caused by Staphylococcus aureus and/or β-hemolytic streptococci group A. This condition is a common cause of ulcerative skin lesions in tropical countries and is often encountered as a secondary infection in travelers. The diagnosis is often made on clinical grounds. Antibacterial treatment for pyoderma should preferably be based on culture outcome. Floxacillin is generally active against S. aureus and β-hemolytic streptococci. Infection with Mycobacterium ulcerans, M. marinum, and M. tuberculosis may cause ulcers. Buruli ulcers, which are caused by M. ulcerans, are endemic in foci in West Africa and have been reported as an imported disease in the Western world. Treatment is generally surgical, although a combination of rifampin (rifampicin) and streptomycin may be effective in the early stage. M. marinum causes occasional ulcerating lesions in humans. Treatment regimens consist of combinations containing clarithromycin, rifampin, or ethambutol. Cutaneous tuberculosis is rare in travelers but may be encountered in immigrants from developing countries. Treatment is with multiple drug regimens consisting of isoniazid, ethambutol, pyrazinamide, and rifampin. Cutaneous diphtheria is still endemic in many tropical countries. Cutaneous diphtheria ulcers are nonspecific and erythromycin and penicillin are both effective antibacterials. Antitoxin should be administered intramuscularly in suspected cases. Anthrax is caused by spore-forming Bacillus anthracis. This infection is still endemic in many tropical countries. Eschar formation, which sloughs and leaves behind a shallow ulcer at the site of inoculation, characterizes cutaneous anthrax. Penicillin and doxycycline are effective antibacterials.Cutaneous leishmaniasis is caused by different species belonging to the genus Leishmania. The disorder is one of the ten most frequent causes of skin diseases in travelers returning from (sub)tropical countries. The clinical picture is diverse, ranging from a painless papule or nodule to an ulcer with or without a scab. Treatment depends on the clinical manifestations and the species involved.Sporotrichosis, chromo(blasto)mycosis, and mycetoma are the most common mycoses that may be accompanied by ulceration. Infections are restricted to certain regions and often result from direct penetration of the fungus into the skin. Anti-mycotic treatment depends on the microorganism involved.The most common causes of infectious skin ulceration encountered in patients from tropical countries who present at a department of tropical dermatology are reviewed in this article.

Serum levels of interferon-γ, tumour necrosis factor-α, soluble interleukin-6R and soluble cell activation markers for monitoring response to treatment of leprosy reactions

Identifying pathogen and host‐related laboratory parameters are essential for the early diagnosis of leprosy reactions. The present study aimed to clarify the validity of measuring the profiles of serum cytokines [interleukin (IL)‐4, IL‐6, IL‐10, interferon (IFN)‐γ and tumour necrosis factor (TNF)‐α], the soluble IL‐6 receptor (sIL‐6R), soluble T cell (sCD27) and macrophage (neopterin) activation markers and Mycobacterium leprae‐specific anti‐PGL‐I IgM antibodies in relation to the leprosy spectrum and reactions. Serum samples from 131 Indonesian leprosy patients (82 non‐reactional leprosy patients and 49 reactional) and 112 healthy controls (HC) from the same endemic region were investigated. Forty‐four (89·8%) of the reactional patients had erythema nodosum leprosum (ENL) while only five (10·2%) had reversal reaction (RR). Follow‐up serum samples after corticosteroid treatment were also obtained from 17 of the patients with ENL and one with RR. A wide variability in cytokine levels was observed in the patient groups. However, IFN‐γ and sIL‐6R were elevated significantly in ENL compared to non‐ENL patients. Levels of IFN‐γ, TNF‐α and sIL‐6R declined significantly upon corticosteroid treatment of ENL. Thus, although the present study suggests limited applicability of serial measurement of IFN‐γ, TNF‐α and sIL‐6R in monitoring treatment efficacy of ENL, reactions it recommends a search for a wider panel of more disease‐specific markers in future studies.

First reported case of Mycobacterium ulcerans infection in a patient from China.

Buruli ulcers have not been previously described in China, and only once at higher latitudes on the northern hemisphere. A patient who travelled in the Shan Dong Province in the Peoples Republic of China developed an ulcer which was proven to be a Buruli ulcer. The clinical picture and histopathological findings from biopsy specimens are characteristic for a Buruli ulcer, and also the growth in culture (Coletsos medium) at a restricted temperature of 30 degrees C. A multiplex polymerase chain reaction (PCR) based on the amplification of the gene encoding for 16S ribosomal RNA and a nested PCR based on the Mycobacterium ulcerans specific repeated sequence 2404 were performed. These PCR investigations identified the bacteria as M. ulcerans, subspecies shinshuense. The patient was initially treated with clarithromycin and rifampicin, which was changed to ciprofloxacin and rifabutin when rifampicin resistance of the first isolate was established. There were no signs of reactivation of the disease 6 months after the end of treatment. M. ulcerans infection occurs above 30 degrees latitude on the northern hemisphere in China and is caused by M. ulcerans, subspecies shinshuense. This case appears to be cured by chemotherapy alone, in contrast to the general experience that surgical treatment is indicated. The granulomatous reaction with only fragments of acid-fast bacteria in the biopsy at the end of treatment many indicate the development of an adequate cell-mediated immune response leading to resistance to the infection.

Cutaneous Leishmaniasis (Leishmania major Infection) in Dutch Troops Deployed in Northern Afghanistan: Epidemiology, Clinical Aspects, and Treatment

Cutaneous leishmaniasis caused by Leishmania major infection affected 172 (18.3%) of 938 Dutch military troops deployed in northern Afghanistan in 2005. The high attack rate was a result of initial insufficient availability of means of prevention and insufficient adherence to preventive measures. At presentation, the lymphatic system was involved in 24.8%. Treatment with intralesional injections of antimony with or without cryotherapy was satisfactory, but 19.5% of patients received secondary treatment with miltefosine. Six months after treatment, 128 (77.1%) of 166 treated patients were cured, 16 (9.6%) were lost to follow-up, and 22 (13.3%) already experienced cure at six weeks but were not seen at six months. Natural evolution played a role in this observational study, which showed cure of all patients seen at six months. In general, management of cutaneous leishmaniasis was feasible under field conditions.

Dipstick assay to identify leprosy patients who have an increased risk of relapse

Classification of leprosy patients into paucibacillary (PB) and multibacillary (MB) determines the duration of treatment; misclassification increases the risk of relapse because of insufficient treatment if an MB patient is classified as PB. We explored the possibility of using a simple dipstick assay based on the detection of antibodies to the Mycobacterium leprae‐specific phenolic glycolipid‐I (PGL‐I) as a tool for classification of patients into PB and MB for treatment purposes. The sensitivity of the dipstick test for detection of MB patients was 85.1%, the specificity 77.7%. We found that of the 71 dipstick negative PB patients 25 (35.2%) were clinically cured at the end of treatment, compared with only two (9.5%) of the 21 dipstick positive PB patients. Of 170 patients in the study population, nine (5.3%) relapsed within the 5‐year follow‐up period. Seven were MB patients, all dipstick positive. Two PB patients relapsed, one was dipstick negative and one was dipstick positive. Dipstick positivity is a risk factor for the future development of relapses, especially in those groups of patients who had received a shorter‐than‐usual course of treatment and the dipstick can be used as an additional, simple tool for classification of patients and for identification of those patients who have an increased risk of relapse.

Immunohistological analysis of in situ expression of mycobacterial antigens in skin lesions of leprosy patients across the histopathological spectrum. Association of Mycobacterial lipoarabinomannan (LAM) and Mycobacterium leprae phenolic glycolipid-I (PGL-I) with leprosy reactions.

The presence of mycobacterial antigens in leprosy skin lesions was studied by immunohistological methods using monoclonal antibodies (MAbs) to Mycobacterium leprae-specific phenolic glycolipid I (PGL-I) and to cross-reactive mycobacterial antigens of 36 kd, 65 kd, and lipoarabinomannan (LAM). The staining patterns with MAb to 36 kd and 65 kd were heterogeneous and were also seen in the lesions of other skin diseases. The in situ staining of PGL-I and LAM was seen only in leprosy. Both antigens were abundantly present in infiltrating macrophages in the lesions of untreated multibacillary (MB) patients, whereas only PGL-I was occasionally seen in scattered macrophages in untreated paucibacillary lesions. During treatment, clearance of PGL-I from granulomas in MB lesions occurred before that of LAM, although the former persisted in scattered macrophages in some treated patients. This persistence of PGL-I in the lesions paralleled high serum anti-PGL-I antibody titers but was not indicative for the presence of viable bacilli in the lesions. Interestingly, we also observed a differential expression pattern of PGL-I and LAM in the lesions of MB patients with reactions during the course of the disease as compared with those without reactions. In conclusion, the in situ expression pattern of PGL-I and LAM in MB patients may assist in early diagnosis of reactions versus relapse.