Mycobacterium avium Complex Infection and AIDS: Advances in Theory and Practice

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1 7 AIDS COMMENTARY Mycobacterium avium Complex Infection and AIDS: Advances in Theory and Practice Constance A. Benson and Jerrold J. Ellner From Rush Medical College, Chicago, Illinois; and Case Western Reserve University School of Medicine, Cleveland, Ohio The role of disseminated infection with the Mycobacterium avium complex (MAC) in the natural history of AIDS has been controversial. It is now clear that this complication of immunosuppression induced by human immunodeficiency virus type 1 (HIV-1) has a major impact upon the quality of life and duration of survival of patients with advanced HIV-1 infection. Progress has been made in our understanding of the bacteriology, pathogenesis, prevention, and treatment of MAC infection. Drs. Constance Benson and Jerrold Ellner (of Rush Medical College in Chicago and Case Western Reserve University in Cleveland, respectively) have led the effort by the AIDS Clinical Trials Group to develop new methods of managing this serious complication of advanced HIV-1 infection. In this AIDS commentary they review our current knowledge of MAC organisms and the clinical problems resulting from infection with these mycobacteria. Disseminated infection due to the Mycobacterium avium complex (MAC) remains the most common systemic bacterial infection among patients with AIDS in the United States, occurring in up to 43% of these individuals [ 1, 2]. Disseminated MAC infection (DMAC) contributes substantially to morbidity and mortality in this population. Since an earlier review of this topic [2], significant advances have been made in basic research on MAC organisms as well as in the treatment and prevention of disease due to these bacteria in patients with AIDS. These advances include a more-complete understanding of the epidemiology, pathogenesis, and natural history of MAC infection; the completion of prospective, randomized trials of single-agent and multidrug treatment; the recognition of the efficacy of macrolides for the treatment of DMAC; and the recent approval by the U.S. Food and Drug Administration (FDA) of rifabutin for the prevention of MAC bacteremia in patients with AIDS and advanced immunosuppression. To place these new data in Received 30 March Grant support: This work was supported in part by NIH (National Institutes of Health) AI (C. A. B.) and by NIH AI , NIH AI , and NIH AI AI (J. J. E. ). Reprints or correspondence: Dr. Constance A. Benson, Rush Medical College/Rush-Presbyterian-St. Luke's Medical Center, 600 South Paulina, Suite 143 AcFac, Chicago, Illinois Clinical Infectious Diseases 1993;17: by The University of Chicago. All rights reserved /93/ $02.00 John P. Phair proper perspective, a review of our knowledge and an update on recent progress are warranted. Microbiology The predominance of M. avium specifically serovars 1, 4, and 8 as a cause of DMAC in patients with AIDS remains constant, and the prevalence of M. avium infection is increasing [1-3]. Recent data bear out the distribution and potential sources of MAC organisms in the environment and the relative virulence of environmental isolates for patients with AIDS. These mycobacteria are ubiquitous and are frequently isolated from water, soil, and foodstuffs. For example, MAC organisms were recovered from 57% of potting soil samples from the homes of patients infected with human immunodeficiency virus type 1 (HIV-1) in San Francisco; 58% of the isolates were identified as M. avium by DNA probing [4]. An interesting discrepancy was found during studies in Uganda: although DMAC among patients with AIDS is extremely rare in that country, M. avium was isolated in abundance from environmental sites [5]. Specifically, M. avium was not found in the blood of 45 patients with advanced AIDS, nor was it represented among 165 respiratory isolates of mycobacteria. Nonetheless, six of seven soil samples contained M. avium (mean, 3,900 cfu/ml), as did four of seven water samples (mean, 420 cfu/ml). The absence of DMAC in patients with AIDS in Uganda is not easily understood. It

2 8 Benson and Ellner CID 1993;17 (July) is possible that the organisms from soil are not fully virulent for patients or that their spread to patients is linked to advanced technological status (e.g., relative to water-purification or hot-water systems). In support of the former possibility, Crowle et al. reported that the replication of M. avit1111 and Mi'cobacteriuin intracellulare was comparable in monocytes from healthy subjects. However, isolates of M. avium replicated more quickly than did environmental isolates of M. intracelltdare within human monocytes from patients with AIDS [6]. The selective growth of M. avium was due primarily to a macrophage defect, although serum from patients with AIDS also promoted the growth of M. avium in macrophages from healthy individuals. New microbiological techniques such as pulsed-field gel electrophoresis (PFGE) and restriction-fragment-length polymorphism (RFLP) analysis allow the genetic characterization of isolates of M. avium and can be applied to the evaluation of the epidemiology and natural history of M. avium infection [7-9]. For example, the use of PFGE in a study of 15 HIV-infected patients coinfected with M. avium revealed that the bacterial strain recovered from each patient was unique [8]. Two of 13 patients evaluated had bacteremia involving two genetically distinct strains. Moreover, two distinct strains were recovered from one of six stool specimens. For each of 10 patients with M. avium isolated from more than one site, all isolates were identical. In other studies RFLP analysis revealed that American and European isolates of M. avium were genetically similar to each other and distinct from African isolates [9]. Epidemiology The epidemiology of DMAC in patients with AIDS has been further clarified by recently completed retrospective and prospective analyses. A retrospective review of the records of 972 AIDS patients at Grady Memorial Hospital in Atlanta showed that, from to , DMAC diagnosed by positive cultures of blood, bone marrow, or liver tissue (obtained by biopsy) increased in incidence from 5.7% to 23.3% [3]. Incidence rates did not differ significantly by gender or risk factor for HIV disease, although there was a trend toward higher rates among younger HIV-infected individuals. This study emphasized the relation between advanced immunosuppression and development of DMAC. A prospective analysis of 159 patients newly referred to the AIDS clinic at this hospital in 1990 revealed a prevalence of DMAC (defined by a single positive blood culture) of 9.6%; MAC was found exclusively in patients with CD4 + lymphocyte counts of <100/mm 3 [3]. The increased incidence of DMAC described among patients with AIDS can be attributed to two factors: (1) greater surveillance related to the recognition ofm. avium as a cause of morbidity and of the improved outcome of therapy reported in recent studies; and (2) longer survival of patients with AIDS as a consequence of the broad use of antiretroviral therapy and of aggressive preventive and interventional treatment of opportunistic diseases. Certainly, more patients with AIDS now survive to the point at which their CD4 + lymphocyte counts fall below 100/mm 3. In another large-scale prospective study of DMAC conducted at Parkland Memorial Hospital in Dallas, monthly blood samples were obtained for culture from 1,006 HIV-infected patients who were followed from the date of AIDS diagnosis [1]. The incidence of MAC bacteremia was 21% at 1 year and 43% at 2 years. The development of MAC bacteremia was associated with a low CD4 + lymphocyte count but was not associated with the patient's age, gender, or race. The incidence of MAC bacteremia 1 year after the diagnosis of AIDS was 39% among patients with CD4 + lymphocyte counts of < 10/mm 3, 15% among those with counts of 40 59/mm 3, and 3% among those with counts of /mm 3 [ 1]. The patients included in this study generally were not given antiretroviral drugs. Chaisson et al. examined the incidence and natural history of MAC disease in a multicenter observational study of patients with HIV disease treated with zidovudine [10]. Data were obtained for 1,020 patients who had AIDS or AIDS-related complex and CD4 + lymphocyte counts of <250/mm 3 at enrollment, who were treated with zidovudine, and who were then followed for 1 year. The incidence of MAC infection was 12%, with a 2-year actuarial risk of 19%. Risk factors for the development of MAC infection included Pnewnocystis carinii pneumonia as the AIDS-defining diagnosis and a relatively low CD4 + lymphocyte count, white blood cell count, and hematocrit at enrollment. Further, each of three factors a CD4 + lymphocyte count of< 100/mm 3, the occurrence of anemia or Pneumocystis carinii pneumonia during follow-up, or the interruption of zidovudine administration was associated with an increased risk of development of DMAC. Pathogenesis Microbial Virulence and Host Defense In light of accumulating evidence that host cytokines may contribute to intracellular parasitism by MAC organisms, subversion of the host defenses appears to be an important factor in the pathogenesis of infection with these bacteria. Substantial progress has been made in defining the impact of a variety of abnormalities in the host's response on the pathogenesis of MAC disease. Colonial morphotypes and pathogenicity. Clinical isolates from patients with DMAC often are mixtures of different colonial phenotypes. Smooth-transparent (SmT) colonial morphotypes ofm. avium show greater potential for intracellular replication and greater virulence in animal models than do isogenic smooth-domed (SmD) organisms [11, 12].

3 CID 1993;17 (July) M. avitlincomplex Infection and AIDS 9 The basis for the morphological difference between SmT and SmD organisms resides in the expression of glycopeptidolipids (GPLs). In chronically infected mice, mycobacteria within phagosomes are surrounded by a multilamellar electron-translucent zone that structurally resembles GPLs and serves as a barrier to intracellular killing [13]. Preliminary evidence suggests qualitative and quantitative differences exist between GPLs from SmT strains and those from SmD strains. Moreover, isolated GPLs from the two morphotypes show differences in cytokine induction that parallel findings with the intact organisms (H. Shiratsuchi and J. J. Ellner, unpublished observations). On the other hand, rough mutants of M. avium also may display virulence; in this case the GPL is not the primary factor associated with intracellular survival [14]. A switch from the SmT to the SmD morphotype occurs with a frequency of during in vitro passage of M. avium [14]. It is not yet clear whether loss of a plasmid or a transposable element is responsible for the switch. The SmD-to-SmT transition also takes place but at a lower frequency ( ). As there is a potential attendant downgrading in the virulence of the isolate with the morphotype switch, the basis for this conversion deserves further study. Infection of human monocytes with M. avium induces the production of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF) a, interleukin (IL) 6, and transforming growth factor (TGF) f3 [15-17]. SmD and SmT colonial morphotypes differ in their ability to induce various cytokines. SmD organisms induce more IL-1 and TNF-a [18-20]; this property may contribute to their lesser virulence since TNF-a possesses macrophage-activating factor (MAF) activity [17]. A possibly related observation is the donor-to-donor variation in the survival of human monocytes infected with M. avium [16]. Survival of monocytes has been associated with increased production of TNF and IL-6. Growth-promoting cytokines and MAFs. The host-parasite balance is complicated further by the apparent ability of certain cytokines to promote both the intracellular and the extracellular growth of M. avilli71 [21, 22]. Both IL-1 a and IL-6 promote the growth of this organism; in fact, M. avium has a receptor for IL-6 [21, 22]. Conversely, a number of cytokines, through a complex network of interactions, inhibit the replication of M. avi Migration-inhibition factor, the first cytokine to be described, has recently been associated with dramatic MAF activity against M. avium [23]. With or without IL-2, both TNF and GM-CSF also have MAF activity against M. avium [15, 17]. The lack of consistency among reports published by different investigators on the MAF activity of interferon (IFN) 7 and other cytokines may relate to the characteristics of the strain of MAC studied and to recently reviewed technological differences in assay procedures [24]. In addition, neutralizing antibody to TGF-/3 unmasks the MAF activity of IFN-7 [25]. Virulent strains of MAC induce greater expression of TGF-0 than do avirulent strains. (The opposite is true for the stimulation of TNF and IL-1 by these morphotypes.) Another example of cytokine antagonism is the blockade of the effector function of macrophages against M. avium by IL-6, apparently via down-regulation of membrane receptors of TNF-a [26]. Therefore, whereas some cytokines have MAF activity or are growth enhancing, others may deactivate macrophages or block the action of MAFs. Other virulence properties of MAC organisms. In addition to differential induction of cytokines, other properties of MAC organisms may be associated with virulence. For example, macrophage vesicles containing live M. avium fail to undergo the usual acidification [27]. This abnormality may offer a survival advantage to the intracellular bacteria by blunting the action of acid cathepsins contained in vesicles, but it may also enhance the intracellular action of azalides, which are very sensitive to low ph. Inhibition of phagosomelysosome fusion also has been noted after the ingestion of virulent M. avium by murine bone marrow-derived macrophages and has been associated with intracellular replication [28]. Role oflymphoc rtes and serum in host defense. As DMAC usually develops late in the course of immunodeficiency, at a time when CD4+ lymphocyte counts are <100/mm 3, it is likely that CD4 +-dependent mechanisms are relatively effective in containing M. avi1,1111 infection. Moreover, the previously described interplay of cytokines is germane to the pathogenesis of disease in a setting in which the number of CD4+ cells is negligible. The potential role of natural killer (NK)-like cells in activating macrophages to kill M. avium is of some interest, given that these cells are CD4 - and may be relatively spared by HIV infection. In vivo depletion of NK activity, in fact, enhances replication ofm. avium in mice [29]. The apparent importance of NK cells parallels the known susceptibility of beige mice (which are NK deficient) to MAC infection. In vitro studies further indicate a potential role of IL-2-activated NK cells in stimulating macrophages through a TNF-a mechanism or by NK production of GM-CSF [15, 30]. The nature of CD4 ±-dependent macrophage activation is not well studied and ultimately may be less relevant because of the severe CD4 + depletion that is associated with the propensity to develop DMAC. It is of some interest, nonetheless, that natural resistance to M. avium may be related to the expression of genes associated with an autoimmune diathesis in New Zealand mice [31]. The role of serum factors in the development of or protection from DMAC is unknown. Preliminary data generated by Crowle and co-workers require confirmation; these authors identified a factor in serum from healthy subjects that inhibits intracellular replication ofm. avium and is absent from the serum of patients with AIDS [32, 33]. Phagocytosis of M. avium by mononuclear phagocytes.

4 10 Benson and Ellner CID 1993;17 (July) Phagocytosis is a necessary first step in defense of the host against MAC organisms. As the bacteria are facultative intracellular parasites, factors that impair phagocytosis actually may favor the host. The alternative complement pathway is essential to the uptake of MAC organisms by mononuclear phagocytes [34]. In fact, CR3 (monocyte surface) receptors, fibronectin receptors, and mannosyl-fucosyl receptors are relevant to MAC uptake [35]. M. avium also appears to bind to extracellular matrix proteins (fibronectin, laminin, and collagen type I) through a fi-1 integrin like molecule on its surface [36]. This surface constituent may play a role in the initial colonization of host epithelial surfaces with M. avium. Cytokines such as IFN-7 block phagocytosis of M. avium, presumably by down-regulating CR3 receptors [37]. As has been noted, this action may block intracellular parasitism and retard the progression of infection. HIV-1 and intracellular growth of M. avium. Clues have recently emerged as to the basis for the remarkable propensity of patients with AIDS to develop DMAC and for the heavy burden of organisms found in tissues in this setting. In one study, monocytes from HIV-infected persons exhibited a normal capacity to control intracellular replication of multiple strains of MAC organisms [38]. In a study by Crowle and co-workers, however, a serum defect was associated with an increased capacity for intracellular replication of MAC organisms in monocyte-derived macrophages from patients with AIDS [33]. HIV infection in vitro promoted the intracellular growth of M. avium when cultivation was prolonged for 14 days [39]. This effect was not apparent during shorter periods of cultivation [39]. Studies of concurrent HIV and mycobacterial infection of macrophages in vitro have revealed no reciprocal effect; however, HIV constituents such as the gp120 envelope protein may directly stimulate intracellular growth of M. avium [40, 41]. Colonization and Dissemination of M. avium It now appears that both the respiratory tract and the gastrointestinal tract are potential sites for dissemination of MAC organisms. A retrospective study of MAC colonization of the respiratory tract was conducted in San Francisco [42]. Thirty-four patients had a culture of respiratory secretions that was positive for M. avium and a subsequent culture of normally sterile fluids that was negative. Twenty-two of these 34 patients later developed DMAC; as the median interval between respiratory isolation and the last negative culture was only 82 days for patients whose infection failed to disseminate, the 65% predictive value ofcolonization presumably is a minimal estimate. The median CD4 + lymphocyte count was 10/mm 3 for colonized patients with subsequent DMAC and 84/mm 3 for individuals without evidence of DMAC. It must be emphasized that respiratory colonization with MAC organisms was associated with low CD4 + lymphocyte counts and that respiratory colonization in the setting of an extremely low CD4 + lymphocyte count was highly predictive of dissemination. In a prospective study of 160 HIV-infected persons with CD4 + lymphocyte counts of <50/mm 3 [43], blood, stool, and induced sputum were cultured at enrollment and at 3- month intervals thereafter. Over a 10-month period, 72% of patients with and 18% of those without a prior positive sputum culture developed DMAC, while 60% of patients with and 1 8% without a prior positive stool culture developed DMAC. The median time to dissemination was 8 months after respiratory colonization and 6.2 months after gastrointestinal colonization [43]. Although respiratory or gastrointestinal colonization was highly predictive of subsequent dissemination, 62% of patients with DMAC had no demonstrable prior colonization. It appears, therefore, that colonization of either the respiratory or the gastrointestinal tract may be a harbinger of but does not uniformly precede dissemination. In three animal models, MAC organisms disseminated after colonization of the gastrointestinal tract. First, intracecal inoculation of 10 6 M. avium cells into mice rendered immunodeficient by thymectomy and CD4 + depletion or by infection with murine AIDS virus (LP-BM5 or Du5H) led to dissemination of the bacteria that was accelerated and enhanced over that in immunocompetent animals [44]. Second, in the beige mouse model, oral administration of MAC organisms led to dissemination of disease [45]. One interesting finding was an association of the ingestion of ethanol with the recovery of increased numbers of viable MAC organisms from the liver. Third, male Sprague-Dawley rats immunosuppressed with cyclosporine developed DMAC after oral challenge with as few as 106 MAC organisms [46]; continuous mycobacteremia developed if the organism load in the spleen exceeded 10'. These models may provide further insights into the pathogenesis of DMAC and should be useful in evaluating approaches to prevention and treatment. In most studies to date, a single positive blood culture has been taken as diagnostic of dissemination. It appears, however, that MAC bacteremia can be intermittent; in a recent study, seven (12%) of 60 patients with a positive screening blood culture subsequently had a negative baseline blood culture before the start of antimycobacterial therapy [47]. Six of these seven patients later had another positive blood culture. Intermittent bacteremia was associated with a shorter period of fever and lower serum levels of alkaline phosphatase at enrollment than was persistent bacteremia. This phenomenon of intermittently positive blood cultures may reflect relatively light burdens of MAC organisms in tissues. Natural History The previously described studies have clarified an emerging pattern of the progression of MAC disease in patients with AIDS: environmental exposure to and acquisition of

5 CID 1993;17 (July) M. avium Complex Infection and AIDS 11 Table 1. Site and frequency of isolation of MAC organisms from patients with AIDS and disseminated MAC infection. Site No. of patients with site sampled Percentage of patients positive Blood Bone marrow Respiratory tract Liver Stool* Gastrointestinal tract Lymph node Urine Other tissuest CSF/brain NOTE. Data are pooled from [48-69]; not all sites were sampled in all cases, and the methods used for recovery of MAC organisms differed among studies. * In some studies, only acid-fast bacillary smears were performed; in others, cultures for MAC organisms were done. t Included were soft-tissue or intraabdominal abscesses, pericardium, and tissues obtained by bone biopsy, joint aspiration, and skin biopsy. MAC organisms; colonization and/or direct invasion of tissues; increasing mycobacterial replication followed by intermittent bacteremia; continuous bacteremia, usually accompanied by clinical symptoms; and, finally, death in the face of advanced immunosuppression. Clinical Presentation A review of prospective studies, published series, and case reports indicates that more than 90% of patients with AIDS and symptomatic MAC infection have evidence of disseminated mycobacterial disease affecting multiple organs and accompanied by continuous, generally high-grade mycobacteremia; MAC organisms may be recovered from other normally sterile tissues in addition to blood (table 1) [48-69]. Autopsy reports indicate that MAC organisms have been found infiltrating virtually every organ system and tissue [70-72]. Localized disease due to MAC is seen less often; entities described in case reports include pulmonary nodules, diffuse infiltrates, cavities or endobronchial lesions, pericarditis, intraabdominal or soft-tissue abscesses, skin lesions, cervical lymphadenitis, osteomyelitis, and parenchymal brain lesions ([55, 59, 61, 66, 73-75] and C. A. Benson, unpublished observations). The most frequently described symptoms, physical findings, and laboratory abnormalities in DMAC are summarized in table 2 [48-50, 54, 61-67, 69]. Symptoms and signs reported for adults have remained relatively consistent among published series and have included fever, night sweats, weight loss or wasting, diarrhea, and abdominal pain. Children with DMAC appear to have similar symptoms [76, 77]. Havlik et al. found that fever, weight loss, and diarrhea Table 2. Frequency of symptoms, physical findings, and laboratory abnormalities among patients with disseminated MAC infection. Finding No. of patients evaluated Percentage of patients positive Fever Night sweats Diarrhea Abdominal pain Nausea/vomiting Weight loss Lymphadenopathy Intraabdominal Mediastinal Hepatosplenomegaly Anemia (<8.5 g of hemoglobin/dl) Elevated serum level of alkaline phosphatase NOTE. Data are pooled from [48-50, 54, 61-67, 69]; not all reports described all symptoms, signs, and laboratory abnormalities or specified the number of patients with each. were statistically associated with DMAC in a subset of prospectively followed patients with AIDS, whereas night sweats occurred with equal frequency among patients with DMAC and those without [3]. The physical findings most commonly reported have included fever, weight loss, intraabdominal lymphadenopathy (as detected by abdominal ultrasonography or computed tomography), and hepatosplenomegaly. The laboratory abnormalities most often identified are anemia usually severe (<8.5 g of hemoglobin/dl) and elevated levels of alkaline phosphatase in serum. Havlik Table 3. Survival trends for patients with AIDS, with or without disseminated MAC infection (DMAC). Median survival period (d) Without After DMAC After DMAC Reference DMAC diagnosis treatment [78]* [79] [42] [ 1 ] [80] NOTE. Ellipses indicate data not collected. * In this study DMAC treatment was defined as at least three antimycobacterial drugs for at least 3 months. The median survival period was calculated in months: it was 11 months long in the absence of DMAC, 4 months long after diagnosis of DMAC, and 8 months long after treatment of DMAC. t The survival period was 190 days long for patients who received a multidrug antimyocbacterial regimen not including clarithromycin and 310 days long for those who received a regimen including clarithromycin.

6 12 Benson and Ellner CID 1993;17 (July) et al. confirmed that both of these abnormalities were statistically associated with DMAC [3]. Whereas few studies to date have correlated specific clinical manifestations of DMAC with prognosis, a number of investigators have reported that patients with AIDS and DMAC survive for a shorter interval than do AIDS patients without DMAC. Table 3 summarizes data from five studies (all retrospective or case-controlled) evaluating the impact of DMAC on the length of the survival period [ 1, 42, 78-80]. The duration of survival after the diagnosis of DMAC appears to have been remarkably consistent among these studies. Sathe et al. showed that anemia was a significant independent negative predictor of the survival of patients with DMAC [79]. Chaisson et al. reported that the development of MAC disease was independently associated with an increased risk of death within a 2-year observation period among patients treated with zidovudine [10]. Some studies suggest that treatment for DMAC may be associated with longer survival; however, the influence of treatment on survival has not yet been evaluated in a prospective, randomized clinical trial. Studies such as these clearly indicate an association between DMAC and a shorter survival interval. Mortality directly attributable to DMAC has been more difficult to establish. Early autopsy studies suggested that MAC infection was rarely a cause of death; more recent postmortem analyses are not available [69-71]. Clearly, MAC infection in most patients does not produce a rapidly or uniformly fatal illness. Rather, the disease is most often of insidious onset and progression, with physiological abnormalities that induce clinical symptoms contributing to a diminished quality of life and that apparently reduce the duration of survival. For those few patients described in published series who were thought to have died of DMAC, the most immediate reason for death appears to have been an overwhelming tissue burden of MAC organisms, with severe inanition, wasting, and attendant organ dysfunction. Diagnosis Isolation of the organism from blood is the most reliable method for the diagnosis of DMAC in patients with AIDS. Blood and bone marrow are the sites from which MAC organisms have been most frequently recovered in most published series (table 1) [48-69]. As has been previously described, these organisms are also recovered often from nonsterile mucosal surfaces, such as those of the respiratory and gastrointestinal tracts, and from normally sterile tissues, such as liver and lymph nodes, in patients with multiorgan disease. Whereas a single positive blood culture is considered diagnostic of disseminated disease, cultures may be only intermittently positive at very low levels of mycobacteremia; if left untreated, most patients with such low-level bloodstream infection will develop sustained bacteremia [47]. For patients producing sputum, the detection of acid-fast bacilli on smears is more likely to represent coinfection with Mycobacterium tuberculosis than that with MAC organisms, and treatment should target the former entity pending culture results. As previously discussed, a culture of respiratory secretions yielding MAC organisms is not diagnostic ofdisseminated disease. Stool cultures are less sensitive than blood cultures for the detection of mycobacteria; this lower degree of sensitivity may be due in part to the decontamination procedures necessary for cultivation of the organism from stool. Acid-fast bacillary smears from stool specimens may be less sensitive than culture; a positive stool smear may reflect a higher bacterial load and may represent disseminated disease [81-83]. Isolation of MAC organisms from bone marrow or other normally sterile tissues may precede recovery of the organisms from the blood; recovery from such sites should be interpreted as indicative of disseminated disease. Mycobacteria can be recovered from blood by inoculation of an unprocessed sample directly into BACTEC 13A (Becton-Dickinson, Towson, MD) or another appropriate broth medium. Alternatively, blood collected in a tube containing an anticoagulant (e.g., sodium polyanetholesulfonate) or in an Isolator tube (Wampole Laboratories, Cranbury, NJ) can be processed and the sediment inoculated onto Middlebrook 7H 10 or 7H11 solid agar (Difco, Detroit) or into BACTEC 12B broth medium [84-87]. The number of colony-forming units of mycobacteria per milliliter cannot be determined by direct inoculation of broth with unprocessed blood; the yield with this technique may be affected by carry-over of antimicrobial agents from the blood sample. In radiometric systems, MAC organisms are commonly detected in broth within 7-14 days, whereas their detection on solid agar medium may require days. With sufficient growth, these organisms can be identified by means of nucleic acid probes within hours, either on agar or in broth medium [87]. The in vitro susceptibility of clinical isolates to antimycobacterial drugs can be tested by standard methods (agar proportion, agar or broth dilution, or radiometric broth dilution). Data on in vitro susceptibility should be interpreted with caution since these results are method dependent and procedures have not been standardized for use in most clinical laboratories. In fact, correlation of in vitro susceptibility with clinical response to treatment of DMAC in patients with AIDS has been demonstrated in clinical trials only for clarithromycin [88]. Treatment Susceptibility of MAC Organisms to Drugs in Vitro, in Macrophages, and in Animal Models The in vitro susceptibility of MAC organisms to a broad spectrum of antimycobacterial agents and the activity of

7 CID 1993;17 (July) M. avium Complex Infection and AIDS 13 various agents against MAC organisms in macrophages and animal models were described in detail in an earlier review [2]. Those agents previously described and currently being evaluated further for their efficacy in the treatment or prevention of DMAC include clarithromycin, azithromycin, rifamycin derivatives, ethambutol, clofazimine, ciprofloxacin, sparfloxacin, amikacin, and liposome-encapsulated gentamicin. Among the most active of these drugs in vitro and in animal models are clarithromycin and azithromycin. Clarithromycin, a macrolide that exerts its antibacterial activity by binding the 50S ribosomal subunit and thereby inhibiting protein synthesis, has an MIC 50 for clinical MAC isolates ranging from <0.25 to 16 yg/ml and an MIC 90 ranging from 0.85 to 16 pg/ml [89-94]. The 14(r)-hydroxyclarithromycin metabolite is 2- to 32-fold less active than the parent compound. Combinations of clarithromycin with other drugs most often have an additive or indifferent effect; synergy or antagonism is observed only rarely. In a study by Heifets et al., MICs and MBCs of clarithromycin were determined for 49 MAC strains from patients with AIDS [93]. The level of inhibitory activity depended on the ph of the medium and the method used for assessment. The drug was more active at ph 7.4 and less active at ph 5.0. The brothdetermined MICs at ph 7.4 were 0.25 and 0.5 ps/ml for most strains. The agar-determined MICs for most strains ranged from 1.0 to 4.0 Ag/mL. The MBCs of the drug were 8- to 64-fold higher than the MICs. The frequency with which clarithromycin-resistant MAC mutants can be induced has been calculated to be >1.8 X 10" [90]. After exposure to clarithromycin alone for up to 16 weeks, the frequency with which resistant mutants were recovered from beige mice infected with MAC organisms ranged from to 10' 77 [95]. Clarithromycin penetrates macrophages; when tested as a single agent at concentrations of 2-4,ug/mL, this drug has been shown to inhibit the replication of MAC organisms in macrophage cell lines [96-99]. Yajko et al. tested the intracellular activity of clarithromycin alone and in combination regimens against MAC strains in the J774 cell line and in alveolar macrophages from HIV-1-infected individuals [99]. Clarithromycin alone reduced the intracellular survival rate of multiple strains of MAC organisms in J774 macrophages; the combinations of clarithromycin plus ethambutol and clarithromycin plus ethambutol and rifampin were mycobactericidal for all strains tested (mean survival rates, 30% and 13%, respectively). Results were similar in studies with alveolar macrophages. In beige mice infected with MAC organisms, clarithromycin either alone or in combination with rifamycins, ethambutol, or clofazimine reduces mycobacterial colony counts in blood, lung, and splenic tissue. The bactericidal activities of seven drugs alone and in combination with clarithromycin were recently compared with the activity of clarithromycin alone in the beige mouse model [100]. When mean pretreatment log ic, numbers of MAC colony-forming units in bone marrow, spleen, liver, and lung were compared with numbers at the end of 4 weeks of treatment, the combinations of clarithromycin plus minocycline and clofazimine and of clarithromycin plus ethambutol, rifampin, and clofazimine were found to have greater bactericidal activity than clarithromycin alone, killing log 10 cfu and log ic, cfu, respectively, in tissues or organs. Azithromycin is an azalide antibiotic that differs from erythromycin by a methyl-substituted nitrogen in the macrolide ring [101]. The drug appears to exert its antibacterial activity by mechanisms similar to those documented for clarithromycin. Azithromycin reaches peak levels of only 1.5 ug/ml in serum concentrations too low to inhibit MAC organisms [102]. (The MIC 90 of azithromycin for MAC isolates tested in vitro is 32 Ag/mL [89, 101].) However, the drug reaches high concentrations in tissues, particularly those in the reticuloendothelial system [89, 102]. Like clarithromycin, azithromycin has been shown to inhibit the replication of MAC organisms in macrophages. The activities of azithromycin, clarithromycin, sparfloxacin, and other drugs were evaluated in a human macrophage model [98]. As assessed in comparison with controls on day 7 after inoculation of macrophages with MAC organisms, clarithromycin, sparfloxacin, and azithromycin slowed intracellular replication of both MAC strains tested; sparfloxacin and clarithromycin exhibited the greatest efficacy. The combination of azithromycin plus sparfloxacin was no more effective than sparfloxacin alone. The comparative activities of azithromycin alone and in combination with other antimycobacterial agents and of clarithromycin have also been evaluated in the beige mouse model of DMAC [103]. The activity of azithromycin was similar to that of clarithromycin in this model [103]. Azithromycin plus clofazimine and ethambutol was more effective than azithromycin alone at reducing the number of MAC organisms in the spleen but not in the lungs. Rifabutin had activity similar to that of azithromycin against organisms in the spleen and the lungs. Rifabutin plus azithromycin was more active than either agent alone against organisms in the spleen but not against those in the lungs. For the three MAC isolates tested, there was little correlation between the in vitro MIC and the in vivo level of activity. Kolonoski and colleagues evaluated azithromycin, ethambutol, and sparfloxacin for the treatment of DMAC in beige mice [104]. Azithromycin alone and ethambutol alone reduced MAC counts in blood by 2.33 and 1.39 log 10 cfu/ml, respectively. Neither azithromycin plus ethambutol nor azithromycin plus sparfloxacin was more effective than azithromycin alone. In a study by Brown and colleagues, azithromycin, rifabutin, or rifapentine was used in a cyclosporine-immunosuppressed rat model of DMAC [105]. No bacteremia was detected in treated animals after 1 month or after 2 months.

8 14 Benson and Ellner CID 1993;17 (July) Table 4. Microbiological response to combination and single-agent therapy for Mycobacterium avium bacteremia. Agent(s)* [reference] No. of patients evaluable at week 4 1. Log ic, cfu at baseline Change in log o cfu at week 4 Percentage of patients culture-negative at week 12 Cpfx/Eth/Rif/Amik* [65] Cpfx/Eth/Rif/Clof [69] Rif [109] Clof [109] Eth [109] L-Gm [112] Azm [67] Clm [88] NOTE. Ellipses indicate data not reported. * Cpfx = ciprofloxacin; Eth = ethambutol; Rif = rifampin; Amik = amikacin; Clof = clofazimine; L-Gm = liposomal gentamicin; Azm = azithromycin; and Clm = clarithromycin. t Not all patients continued to receive antimycobacterial therapy through week 12 or had blood obtained for culture at week Geometric mean MAC colony count at baseline was 537 cfu/ml and at week 4 was 14 cfu/ml. Moreover, comparisons with untreated controls revealed significantly fewer mycobacteria in the spleens and livers of treated animals after 32 days and in their lungs after 60 days. Rifabutin cleared MAC organisms from all organs more rapidly than did azithromycin or rifapentine. Preventive strategies have been evaluated in animal models. Administration of azithromycin to beige mice daily for 14 days before challenge with 2.2 X 10 8 MAC organisms reduced the frequency of development of bacteremia; when also administered on days 1, 3, 5, 7, and 9 after challenge, azithromycin reduced the number of viable organisms in liver, appendix, and spleen by 62%-94% from counts in controls [106]. In the cyclosporine-treated rat model, no evidence of DMAC was found 4 months after challenge with 104 cfu of MAC organisms when animals were pretreated with azithromycin, rifabutin, or rifapentine [105]. Bacteremia was documented in four of six control animals after 4 months but did not develop in treated animals. Prospective Clinical Trials of Treatment for DMAC in Patients with AIDS Among the most important advances in the study of DMAC in the. past 2 years is the development of more effective regimens for treatment and prevention. The short-term efficacy of multidrug and single-drug regimens consisting of agents active against MAC organisms in vitro has been demonstrated. Quantitative colony counts of MAC organisms in blood have been employed as surrogate markers of microbiological efficacy in these studies; declines in colony counts have been associated with clinical improvement. Studies of drug combinations. In a prospective study of ethambutol (15 mg/[kg d] for 12 weeks), rifampin (600 mg/ d for 12 weeks), ciprofloxacin (750 mg twice daily for 12 weeks), and amikacin (10 mg/[kg d] for 4 weeks) in 17 patients with AIDS and DMAC, the geometric mean MAC colony counts in blood declined from a baseline value of 537 cfu/ml to 14 cfu/ml at week 4 (table 4) [65]. This microbiological response was sustained for 12 weeks in most cases and was associated with a reduction in clinical symptoms. However, premature discontinuation of therapy was necessary in 41% of cases, mostly because of adverse reactions or intercurrent illnesses. In a similar study, Kemper et al. evaluated the above regimen of ethambutol, rifampin, and ciprofloxacin with clofazimine (100 mg/d) substituted for amikacin in 31 patients with AIDS and MAC bacteremia [69]. Mean counts of MAC organisms in blood declined from a baseline value of 2.1 log 10 cfu/ml to 0.7 log io cfu/ml after 4 weeks (table 4). Forty-two percent of patients had at least one negative blood culture during therapy; the median time to a negative blood culture was 12 weeks. Suppression of MAC bacteremia was sustained during therapy and was associated with a reduction in clinical symptoms; however, 37% of patients were unable to complete 12 weeks of full-dose treatment, largely because of adverse reactions. Jacobson and colleagues compared the combination of ethambutol (25 mg/[kg d]), rifampin (600 mg/d), and ciprofloxacin (750 mg/d) with placebo in a prospective, randomized study of 8 weeks' duration [107]. MAC colony counts in blood declined by >1.0 log io cfu/ml for four of nine patients receiving the multidrug regimen but for none of 10 patients receiving placebo (P ). Dose-limiting toxicity of one or more agents developed in nine of 13 evaluable patients in the treatment group. Seventy-nine patients with AIDS and MAC bacteremia were enrolled in AIDS Clinical Trials Group Protocol 135 a randomized, prospective, comparative trial of ciprofloxa-

9 CID 1993;17 (July) M. aviuni Complex Infection and AIDS 15 cin, ethambutol, rifampin, and clofazimine with or without amikacin. Preliminary analysis showed that amikacin had no great impact on microbiological end points (J. J. Ellner, unpublished observations). In a European study evaluating 12 patients with AIDS and MAC bacteremia, a combination of clarithromycin (1 g twice daily), ciprofloxacin (500 mg three times daily), and amikacin (7.5 mg/kg twice daily) resulted in clearance of bacteremia and resolution of symptoms in all cases after 2-8 weeks of treatment [108]. Amikacin was administered for 3 weeks, while clarithromycin and ciprofloxacin were given for weeks. Eight of the 12 patients enrolled had received antimycobacterial drugs previously (mean, 11.8 weeks before enrollment); however, all such treatment had been discontinued for a minimum of 2 weeks before therapy with the combination was initiated. Adverse gastrointestinal reactions, reported by 25% of patients, were rendered less severe by the reduction or staggering of drug doses. In each of these studies, MAC colony counts in blood declined during therapy; this fall was usually accompanied by clinical improvement. However, as in previous retrospective studies, it was difficult to ascertain the contribution of any single agent to the efficacy of the multidrug regimens administered. Adverse reactions were common in each study, with gastrointestinal intolerance predominating. Single-agent studies. In a double-blind, randomized, placebo-controlled study conducted by Dautzenberg et al., MAC colony counts in blood decreased markedly in seven HIV-infected patients during an initial 6-week period of treatment with clarithromycin (1 g twice daily), and five of the seven patients became culture-negative [68]. Conversely, MAC colony counts increased in four patients during the initial 6 weeks of placebo administration. Kemper and co-workers assessed the effects of ethambutol, rifampin, and clofazimine in 30 patients with AIDS and MAC bacteremia [109]. Patients were randomly assigned to receive ethambutol (15 mg/[kg d]), rifampin (600 mg/d), or clofazimine (200 mg/d) alone for 4 weeks and then crossed over to receive the other two agents in combination for 4 weeks. The median change in MAC colony count after 4 weeks of treatment was 0.6 log in cfu/ml with ethambutol alone, +0.2 log in cfu/ml with rifampin alone, and 0.2 log in cfu/ml with clofazimine alone (table 4). The combination of ethambutol plus rifampin or clofazimine reduced colony counts no more than did ethambutol alone. The efficacy of clarithromycin as a single agent was further clarified in two multicenter, randomized, dose-ranging studies. The first, conducted by Chaisson et al., included 154 patients with AIDS and MAC bacteremia who were randomized to receive 500 mg, 1 g, or 2 g of clarithromycin twice daily for 12 weeks [88]. The median reduction in colony counts from baseline ranged from 99.4% to 100% figures that represented a decline of 2.6 log in cfu/ml at 12 weeks (table 4). The time to clearance of MAC bacteremia was dose dependent, with a median of 55, 43, and 27 days for the 500-mg, 1-g, and 2-g arms, respectively. The microbiological response was accompanied by an alleviation of symptoms. Adverse reactions, primarily gastrointestinal, were dose related. All MAC isolates were initially susceptible to clarithromycin, with MICs of <2 ps/ml. Bacteriologic relapses, usually accompanied by symptoms and associated with increases in MICs of clarithromycin to >4 ktg/ml, were documented in 20% of patients by week 12. In the second study, 299 patients were randomized to receive 500 mg or 1 g of clarithromycin twice daily [110]. A bacteriologic response, defined as a negative blood culture or a decline in colony counts by >1 log in cfu/ml at the end of week 4, was reported for 72% of patients receiving 500-mg doses and for 82% of those receiving 1-g doses. Clinical responses were noted in 75% and 82% of the patients in these respective groups. In a pilot study, monotherapy with azithromycin (500 mg/ d) resulted in a mean reduction in colony counts from 118 to 43 cfu/ml in three patients treated for 10 days and from 2,028 to 136 cfu/ml in 21 patients treated for 20 or 30 days [67]. Among the latter 21 patients, fever and night sweats became less severe. The most common adverse reaction to therapy was diarrhea. In a pilot study of treatment of DMAC with sparfloxacin, Young and colleagues administered oral doses of either 200 mg/d or 300 mg/d for 4 weeks to 22 evaluable patients [111]. Four of 10 patients given 200 mg/d and three of 12 patients given 300 mg/d had a decline in MAC colony counts in blood; the average decrease was by 0.53 log in cfu/ml and 0.96 log in cfu/ml for these two groups, respectively. In the remaining 15 patients, the level of bacteremia remained constant or increased. The response of symptoms to treatment was difficult to assess because of insufficient data. In a dose-escalation study including 21 evaluable patients, liposome-encapsulated gentamicin reduced mean MAC colony counts in blood by >90% at each dose level ( mg/[kg d]) by the end of week 4 [112]. Symptoms were alleviated in patients receiving the highest dose; one of the latter patients developed nonoliguric renal failure. Prophylaxis Two identical, prospective, randomized, double-blind, placebo-controlled clinical trials of rifabutin (300 mg/d) for prophylaxis of MAC bacteremia in patients with AIDS have been completed [ ]. A total of 1,146 patients who had AIDS and CD4 + lymphocyte counts of <200/mm 3 and who were receiving antiretroviral therapy and prophylaxis for P. carinii pneumonia were enrolled in the United States and Canada from February 1990 to January 1992; 590 patients participated in the first study and 556 in the second. In an intent-to-treat analysis, 24 rifabutin recipients and 51 placebo recipients in the first study developed MAC bac-

10 16 Benson and Ellner CID 1993;17 (July) teremia (P =.001); similarly, 24 rifabutin recipients and 51 placebo recipients in the second study developed bloodstream infection with MAC organisms (P =.002). The hazard ratio of placebo to rifabutin for the time to development of MAC bacteremia was 2.33 in the first study (log rank P =.001, Wilcoxon P <.001) and 2.11 in the second (log rank P =.002, Wilcoxon P =.005) [113, 114; data on file, Dublin, OH: Adria Laboratories, 1992]. Analysis of MAC bacteremia free survival revealed a hazard ratio of placebo to rifabutin of2.739 in the first study (log rank P =.001, Wilcoxon P =.006) and of in the second (log rank P <.001, Wilcoxon P =.001) [113, 1 14; data on file, Dublin, OH: Adria Laboratories, 1992]. Overall survival was not statistically different for the two groups in either study; however, in both studies, fever, fatigue, and weight loss were noted earlier in placebo recipients than in rifabutin recipients. Few adverse reactions were attributed to rifabutin. Further analysis indicated that nearly 50% of the 48 patients in the rifabutin treatment groups who developed MAC bacteremia either were not taking or were not absorbing the drug when the bloodstream infection was detected [115]. MICs of rifabutin were no higher for MAC isolates from rifabutin recipients than for those from placebo recipients [114]. On the basis of these analyses, the investigators concluded that daily treatment with rifabutin was safe and that it delayed or prevented the development of MAC bacteremia in patients with AIDS. Summary Recommendations Diagnosis and Treatment of DMAC The data gathered and evaluated so far are sufficient to allow rational recommendations for the management of DMAC in patients with AIDS. The patients at greatest risk for DMAC are those with CD4 + lymphocyte counts below 100/mm 3; in fact, when disseminated disease is detected, the majority of patients have counts below 50/mm 3. Routine screening of patients at risk by means of serial blood cultures for MAC organisms does not appear to be warranted at this time. Recovery of MAC organisms from nonsterile body fluids or tissues (e.g., the respiratory tract or stool) in the absence of symptoms suggests colonization. In this situation patients appear to be at increased risk of dissemination, particularly if their CD4 + lymphocyte count is below 100/mm 3 ; thus they require more intensive monitoring of signs and symptoms. However, the available data do not support routine screening of respiratory secretions or stool for MAC colonization. Whether colonized patients should be treated or should receive more intensive prophylaxis remains to be determined. Recovery of MAC organisms from blood, bone marrow, or normally sterile tissues is sufficient for the diagnosis of DMAC. Intermittent mycobacteremia may be documented and probably reflects a relatively low burden of organisms in tissues; if left untreated, this intermittent syndrome will progress to sustained bacteremia in most instances. Therefore, multiple positive blood cultures are unnecessary. Instead, in the diagnostic evaluation of a febrile or symptomatic HIV-infected patient with advanced immunosuppression, a single blood sample should be cultured for MAC organisms; when patients have persistent symptoms in the absence of an alternative diagnosis, culture of a second blood sample and/or culture or biopsy of bone marrow or of material from other clinically involved sites should improve the diagnostic yield. Ancillary studies, such as acid-fast bacillary smear or culture of stool or abdominal imaging, may help identify such sites. Patients with DMAC should be treated. Treatment clearly is associated with a reduction in mycobacterial load and an alleviation of symptoms, although neither the elicitation of a durable response nor the extension of survival has yet been proved in a prospective clinical trial with prolonged follow-up. The initial regimen administered should include at least two antimycobacterial drugs that may delay or prevent the emergence of resistance. Oral clarithromycin, in a dose of 1 g twice daily, may be the preferred first agent; 500 mg twice daily may be used for patients intolerant of higher doses. Oral azithromycin, in a dose of 500 mg/d to 1 g/d, may be an acceptable alternative first agent, although published data suggest that this drug may be less active than clarithromycin in vitro and clinical experience with its use for this indication is more limited. On the basis of the data presented above, ethambutol seems the most rational choice as a second drug, at a preferred dose of 15 mg/(kg d). The decision about whether to use additional drugs depends in part on the clinical status of the patient. Two or three additional agents may be used for those who are more severely symptomatic or more seriously ill and the intensity of therapy reduced once a clinical and microbiological response is evident. Little comparative information supports the preferential use of one drug over another. A choice from among the rifamycin derivatives (rifampin or rifabutin, 600 mg/d), clofazimine ( mg/d), ciprofloxacin ( mg twice daily), or parenteral amikacin (10 mg/[kg d]) should take into account clinical considerations such as the potential for drug interactions, the tolerability of multiple oral agents associated with adverse gastrointestinal reactions, and the patient's vulnerability to hepatotoxicity or nephrotoxicity. A clinical response, including reductions in fever and systemic symptoms, may require 2-8 weeks, with the precise interval depending on the severity of illness and the mycobacterial load in the tissues or the bloodstream. While quantitative blood cultures may be appropriate for clinical trials, they are not necessary for the routine monitoring of therapy. A repeat blood culture after 4-8 weeks may be useful for assessment of the microbiological response. Therapy should be continued for life.

11 CID 1993;17 (July) M. avium Complex Infection and AIDS 17 Susceptibility of MAC isolates to Ag of clarithromycin/ ml has been shown to correlate with clinical and microbiological response; thus far, this correlation has not been demonstrated for any other agent in the treatment of DMAC in patients with AIDS. When patients fail to respond to initial therapy after 4-8 weeks, it may be reasonable to test isolates for susceptibility to clarithromycin or azithromycin. Recurrent MAC bacteremia and relapse of symptoms have been associated with an MIC of clarithromycin of >8 µg/ml [88]. The optimal drug combinations and their impact on longterm survival have yet to be established in prospective, comparative trials. Prophylaxis of DMAC The data generated in the rifabutin prophylaxis trial were sufficiently compelling to result in FDA approval of this drug for the prevention of MAC bacteremia. That MAC bacteremia can be prevented or delayed in a substantial proportion of patients is encouraging, and those patients at greatest risk-chiefly those with a CD4 + lymphocyte count of <100/ mm 3-should receive prophylaxis. Rifabutin (300 mg/d) is the only agent approved by the FDA for this indication. Rifabutin may not, however, be appropriate for all individuals at risk. Among the major concerns in this regard are (1) the theoretical potential for the selection of resistant isolates of M. tuberculosis in areas where tuberculosis is endemic or in populations whose risk of primary or reactivation tuberculosis is high, (2) the potential for drug interactions that may limit this agent's use, and (3) controversy about the management of patients intolerant to or otherwise unable to take rifabutin. 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