In an effort to find a potential alternative treatment for scrub typhus, we evaluated the effectiveness of the standard drug doxycycline and the new macrolide azithromycin against a doxycycline-susceptible strain (Karp) and a doxycycline-resistant strain (AFSC-4) of Rickettsia tsutsugamushi. The antibiotics were tested in an in vitro assay system in which infected mouse fibroblast cells (L929) were incubated for 3 days in various concentrations of the drugs. Rickettsial growth was evaluated by direct visual counts of rickettsiae in Giemsastained cells or by flow cytometry. Initial tests were conducted at the concentration of each antibiotic considered to be the upper breakpoint for susceptibility (16 g/ml for doxycycline and 8 g/ml for azithromycin). Growth of both Karp and AFSC-4 was strongly inhibited with both antibiotics, as measured by visual counts, although the percentage of cells infected with AFSC-4 in the presence of doxycycline was three times greater than the percentage of cells infected with Karp but was only 60% as great as the percentage of cells infected with Karp in the presence of azithromycin. Flow cytometry confirmed that rickettsial growth occurred in the absence of antibiotics, but it failed to detect it in the presence of high concentrations of either drug. Visual counts of rickettsial growth at lower concentrations of the antibiotics (0.25 to 0.0078 g/ml) showed that the Karp strain was 16 times more susceptible that the AFSC-4 strain to doxycycline. Azithromycin was much more effective than doxycycline against AFSC-4, inhibiting rickettsial growth at 0.0156 g/ml to levels below that achieved by 0.25 g of doxycycline per ml. Azithromycin was also more effective than doxycycline against the Karp strain, causing greater reductions in the number of rickettsiae per cell at lower concentrations. If in vivo testing confirms the in vitro effectiveness of azithromycin, it may prove to be the drug of choice for the treatment of scrub typhus in children and pregnant women, who should not take doxycycline, and in patients with refractory disease from locations where doxycycline-resistant strains of R. tsutsugamushi have been found. When tested in an in vitro assay system, azithromycin was more effective than doxycycline against doxycyclinesusceptible and -resistant strains of R. tsutsugamushi.
Although delayed-type hypersensitivity skin testing with tuberculin purified protein derivative (PPD) is the standard for tuberculosis screening, its variability suggests the need for a more sensitive, noninvasive test. An in vitro whole-blood assay has been proposed as an alternative. Using health care worker volunteers, we confirmed the correlation between PPD skin test (PPD- The immune response to mycobacterial infection is predominantly cellular (5). Delayed-type hypersensitivity (DTH) skin testing has been a convenient, cost-effective method for assessing cell-mediated immune responses to a variety of antigens, starting with the mycobacterium-derived tuberculin purified protein derivative (PPD) over 100 years ago (28). Also known as the Mantoux test, this method has been the "gold standard" for diagnostic screening for detection of new or asymptomatic Mycobacterium tuberculosis infections. Although the test is reasonably priced, there continue to be multiple factors challenging the accuracy of the PPD skin test (PPD-ST) in different settings. These factors include (but are not limited to) special nursing skill requirements for placement and reading, variability in operator placement and reading, cross-reactivity among mycobacterial species (including M. avium and M. bovis BCG), the need for the patient to return in 48 to 72 h for a reading, and the modulation of the skin response due to underlying illness or immunosuppression (6,28). Although the specificity of a significant positive test exceeds 95% in cattle, the sensitivity of the test in both animals and humans may be less than 75% (11, 31).STThe immune response to M. tuberculosis is highly dependent upon gamma interferon (IFN-␥) production by macrophages and antigen-specific T cells (9). Over the past decade, there has been an increasing interest in the development and application of in vitro culture assays measuring IFN-␥ production in response to tuberculin antigen stimulation as diagnostic screening tests substituting for the classic PPD (19). Although it initially used peripheral blood mononuclear cells (PBMC), the methodology evolved to a whole-blood culture technique that was first validated in Australian cattle (24, 31). The wholeblood culture technique requires less incubation time, is technically simpler, and provides a milieu closer to in vivo conditions. A standardized diagnostic kit with a specifically defined data analysis procedure, using human PPD, avian PPD, and the mitogen phytohemagglutinin (PHA), has been marketed by CDL Limited in Australia (QuantiFERON-TB or Q-IFN).The purpose of this study is twofold: first, to assess the agreement between the Q-IFN assay and the Mantoux skin test (PPD) in classically PPD positive and PPD negative healthy volunteers; second, to evaluate five separate fractional extracts derived from M. tuberculosis cultures in the same Q-IFN assay system and determine concordance with the PPD-ST results. MATERIALS AND METHODSParticipants and skin testing. Forty-eight volunteers, all hospital employees, were recrui...
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