We evaluated PCR methods for diagnosis of acute and chronic cutaneous leishmaniasis (CL) in an area of Colombia where Leishmania (Viannia) is endemic. The PCR method specifically amplified whole linearized minicircle kinetoplast DNA (kDNA) of the Leishmania subgenus Viannia from biopsy lysates. PCR products were detected in agarose gels. For 255 acute cases, this PCR method had greater sensitivity (75.7%) than each conventional method, i.e., microscopic examination of Giemsa-stained lesion scraping (46.7%), biopsy culture (55.3%), aspirate culture (46.3%), and the conventional methods combined (70.2%). Among 44 cases of chronic CL, amplification of biopsy DNA was more sensitive (45.5%) than the individual (4.5 to 27.7%) and combined (27.3%) conventional methods. The detection of kDNA in biopsies from chronic lesions was enhanced by a chemiluminescent dot blot hybridization, which produced a sensitivity of 65.8% when alone and 90.9% when in combination with DNA extraction of biopsy lysates (P < 0.001). Three biopsies from 84 skin lesions of other etiologies were falsely positive by PCR (specificity, 96.4%). PCR detected kDNA more frequently in biopsies (detection level, 83.9%) than in aspirates (74.7%) from 103 cases of acute CL. Among aspirates from 53 chronic cases of CL, the alternative methods, DNA extraction and hybridization, increased sensitivity from 41.5 to 56.6% (P > 0.05). This enhanced PCR method in chronic biopsies was so much more sensitive than conventional methods that it should be considered the preferred diagnostic method for chronic CL. These findings support the appropriate incorporation of PCR into diagnostic strategies for cutaneous leishmaniasis.The leishmaniases are a group of illnesses of the skin, oral and respiratory mucosae and the reticuloendothelium caused by protozoa of the genus Leishmania. Of these, the cutaneous form is the most widespread, afflicting primarily rural and periurban populations exposed to the infected sand fly vector. Cutaneous leishmaniasis (CL) is most frequently diagnosed by clinical evaluation, either alone or in combination with the leishmanin skin test. Clinical evaluation usually suffers from lack of standardization (13,38) and is hampered by the fact that even fairly typical acute lesions can be confused with other dermatological problems, such as sporotrichosis (9). The leishmanin skin test is highly sensitive but lacks specificity when employed in areas of endemicity because it cannot distinguish acute lesions from past infection. A definitive, laboratory diagnosis of mucosal or cutaneous leishmaniasis traditionally requires either the visualization of amastigotes or the isolation of replicative Leishmania from lesions (24). The most widely employed laboratory methods for CL are microscopic examination of lesion scrapings, biopsy impression smears, and histopathology. The most sensitive conventional diagnostic methods, culture of lesion biopsies and aspirates, are available only in reference laboratories. Even these less available, more sensitive methods ...

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Luciferase reporter phages (LRPs) have proven to be efficient tools for drug susceptibility testing of Mycobacterium tuberculosis. Luminometric detection of LRP activity offers higher sensitivity and quantitative results, while a Polaroid film detection method offers a "low-tech" inexpensive alternative that is called the Bronx box. In this work we evaluated, improved, and compared the performance of the luminometer and the Bronx box formats for drug susceptibility testing with LRPs by using 51 clinical isolates of M. tuberculosis, with the agar proportion method (PM) serving as reference. The sensitivity in detecting resistance to isoniazid and rifampin, antibiotics that define multidrug resistance (MDR), was 100% for both methods. The turnaround time for results was reduced from 3 weeks for PM to 54 or 94 h for luminometry or the Bronx box, respectively. These results support the utility of LRPs as a screening test for the surveillance of MDR tuberculosis.Early diagnosis of tuberculosis (TB) and drug-resistant TB allows the prescription of appropriate antibiotic regimens, leading to more efficient control of the disease (15). The increase in incidence of multidrug-resistant (MDR) TB (4) has clearly established the need to improve the drug susceptibility techniques available. Mycobacteriophages (phages) are promising tools for the early diagnosis of drug-resistant TB because they offer a phenotype-based result in a turnaround time similar to that of some molecular approaches at a low cost. Luciferase reporter phages (LRPs) are phages harboring the fflux reporter gene, which codes for the firefly luciferase, which in turn catalyzes a reaction that releases light in the presence of its substrate luciferin and ATP. LRPs are able to infect, replicate, and express their genome and the fflux gene only within viable mycobacterial cells. Luciferase activity can then be detected only if cellular ATP is present (8), allowing detection of M. tuberculosis in clinical samples (2, 13). If a decontaminated clinical specimen containing M. tuberculosis is pretreated with antibiotics and is then infected with LRPs, light emission will be proportional to mycobacterial viability; hence, LRPs are promising candidates for drug susceptibility testing (3,12,14). Detection of the luciferase activity is achieved by means of a luminometer or photographic film. The luminometer offers higher sensitivity and quantitative results (1, 2); the Polaroid film offers an inexpensive, "low-tech" alternative that is called the Bronx box (14), but its performance as a clinical tool requires further evaluation.In this work we improve, evaluate, and compare the performance of the luminometer and the Bronx box formats for drug susceptibility with LRPs by using clinical isolates of M. tuberculosis obtained from a focus presenting high levels of resistance, with the agar proportion method (PM) (9) LRPs. Stocks of phages phAE85 (3, 12) and phAE142 (1, 2) were amplified as previously indicated. Titers of phAE85 and phAE142 were 5 ϫ 10 8 and 7.5 ϫ 10 6 PFU/...

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