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Evaluation of the MycoAKT latex agglutination test for rapid diagnosis of Mycobacterium avium complex infections.

Rapid diagnosis of Mycobacterium avium complex (MAC) bacteremia is important for management of patients with the acquired immunodeficiency syndrome who have disseminated MAC. The purpose of this study was to determine the reliability of the MycoAKT latex agglutination test for direct detection of MAC in positive mycobacterial blood cultures. First, colonies of isolates of previously identified mycobacteria, including 35 MAC, were tested. Of the 55 isolates evaluated, 33 were identified as MAC by the latex test, including 31 of the known MAC and 2 M. chelonae (sensitivity, 88.6%; specificity, 90.0%). Second, broth from 20 ESP II and 20 BACTEC 12B bottles seeded with isolates of MAC were tested. Aliquots from 19 (95%) ESP II cultures and 16 (80%) 12B cultures were positive by the latex test. In phase 3, broth from 115 signal-positive ESP II blood cultures were tested by latex agglutination. Forty-three subcultures from these bottles grew mycobacteria (41 MAC and 2 Mycobacterium tuberculosis complex); the remainder grew no organisms. Broth from 40 of the blood cultures (39 that grew MAC and 1 from which no organisms were recovered) were latex positive; thus, the sensitivity, specificity, and positive and negative predictive values of the latex test for direct identification of MAC in ESP II blood cultures were 95.1, 98.6, 97.5, and 97.3%, respectively. The mean time to detection of MAC was 14.6 days (range, 6-34 days) with the direct latex test, compared with 18.3 days (range, 9-36 days) with subculture and probe (p < 0.05).

AIDS-Related Opportunistic Infections↗

Splenectomy in a child with chronic Mycobacterium avium complex infection and splenic sequestration.

Mycobacterium avium complex infections (MAC) are being reported with increasing frequency in immunocompromised patients. When these infections become resistant to standard antibiotic therapy, treatment with interferon-gamma (IFN-gamma) can be helpful. Pain, fever, splenic enlargement, and cytopenias caused by splenic sequestration developed during IFN-gamma treatment in a 9-year-old boy and were successfully treated by splenectomy. The development of IFN-gamma-induced splenic sequestration and cytopenias in MAC-infected patients represents a new indication for splenectomy.

Anti-Bacterial Agents↗

Multiple serovars of Mycobacterium avium complex in patients with AIDS.

Mycobacterium avium complex (MAC) was isolated and serotyped from 127 samples from 43 HIV-infected patients with disseminated disease in Sweden. Thirteen different serovars were observed. Serovar 6 was the most common, followed by 4, 9 and 11. Serovar 8 was rare. In 22 of the patients the same serovar was found in blood and at other sites. Clinical symptoms and outcome were compared in patients with different serovars. Analysis of patient records revealed no association between clinical picture and any specific serovar. The median survival time after MAC infection was 7 months. Somewhat shorter survival was observed in patients with serovar 4 than in those with serovar 6.

Acquired Immunodeficiency Syndrome↗

Correlation of quantitative bone marrow and blood cultures in AIDS patients with disseminated Mycobacterium avium complex infection.

The relationship between Mycobacterium avium complex (MAC) infection of blood and bone marrow was studied in human immunodeficiency virus-infected patients before and during treatment. Quantitative cultures were obtained at baseline from 17 persons with newly detected MAC bacteremia. Serial blood cultures were obtained, and a second bone marrow sample was obtained at 4 or 8 weeks. At baseline, the median MAC load in bone marrow core samples was 3 log10 higher than in blood. Bone marrow MAC loads ranged widely (866-847,315 cfu/g), and no significant correlation was found between MAC load in blood and that in bone marrow core samples. MAC loads in bilateral bone marrow biopsy samples from 7 subjects were highly correlated. MAC loads declined in blood and bone marrow at similar rates during therapy, but blood was sterilized before bone marrow. Length of survival was inversely associated with initial bone marrow core MAC load but not with blood MAC load. Initiation of treatment when tissue MAC load is low may increase the likelihood of favorable clinical outcome.

AIDS-Related Opportunistic Infections↗

Differences in serological responses to specific glycopeptidolipid-core and common lipid antigens in patients with pulmonary disease due to Mycobacterium tuberculosis and Mycobacterium avium complex.

Disease due to the Mycobacterium avium complex (MAC) is one of the most important opportunistic pulmonary infections. Since the clinical features of MAC pulmonary disease and tuberculosis (TB) resemble each other, and the former is often difficult to treat with chemotherapy, early differential diagnosis is desirable. The humoral immune responses to both diseases were compared by a unique multiple-antigen ELISA using mycobacterial species-common and species-specific lipid antigens, including glycopeptidolipid (GPL)-core. The results were assessed for two patient groups hospitalized and diagnosed clinically as having TB or MAC pulmonary disease. Diverse IgG antibody responsiveness was demonstrated against five lipid antigens: (1) monoacyl phosphatidylinositol dimannoside (Ac-PIM2), (2) cord factor (trehalose 6,6'-dimycolate) (TDM-T) and (3) trehalose monomycolate from Mycobacterium bovis Bacillus Calmette-Guérin (BCG) (TMM-T), and (4) trehalose monomycolate (TMM-M) and (5) GPL-core from MAC. Anti-GPL-core IgG antibody was critical, and detected only in the primary and the secondary MAC diseases with high positivity, up to 88.4 %. However, IgG antibodies against Ac-PIM2, TDM-T and TMM-T were elevated in both TB and MAC patients. Anti-TMM-M IgG antibody was also elevated in MAC disease preferentially, with a positive rate of 89.9 %, and therefore, it was also useful for the diagnosis of the disease. IgG antibody levels were increased at the early stages of the disease and declined in parallel to the decrease of bacterial burden to near the normal healthy control level, when the anti-mycobacterial chemotherapy was completed successfully. Unexpectedly, about 25 % of hospitalized TB patient sera were anti-GPL-core IgG antibody positive, although the specificity of GPL-core was sufficiently high (95.8 % negative in healthy controls), suggesting that a considerable number of cases of latent co-infection with MAC may exist in TB patients. Taken together, the combination of multiple-antigen ELISA using mycobacterial lipids, including GPL-core and TMM-M, gives good discrimination between healthy controls and sera from patients with TB or MAC disease, although for accurate diagnosis of TB more specific antigen(s) are needed.

Antibodies, Bacterial↗

A case of Mycobacterium avium complex pulmonary disease in an immunocompetent host.

Mycobacterium avium complex is the most common of nontuberculous mycobacterium capable of causing disease in humans. The manifestations can vary from asymptomatic exposure to hypersensitivity pneumonitis to advanced fibrocavitary disease. We describe the case of a 37-year-old male with advanced cavitary disease from Mycobacterium avium complex. The patient had no previous history of pulmonary disease, but did have a history of snorting crushed Lortab (hydrocodone/acetaminophen) tablets and we postulate that possible injury to the lung from talc or other chemicals in the tablets may have predisposed him to develop Mycobacterium avium pulmonary disease.

Adult↗

Evaluation of a commercial line probe assay for identification of mycobacterium species from liquid and solid culture.

The performance of a commercial line probe assay (LiPA) (Inno-LiPA Mycobacteria; Innogenetics, Belgium) for the detection and identification of Mycobacterium species from liquid and solid culture was evaluated at five routine clinical laboratories. The LiPA method is based on the reverse hybridization principle, in which the mycobacterial 16S-23S ribosomal RNA (rRNA) spacer region is amplified by polymerase chain reaction (PCR). Amplicons are subsequently hybridized with oligonucleotide probes arranged on a membrane strip and detected by a colorimetric system. The test detects the presence of Mycobacterium species and specifically identifies Mycobacterium tuberculosis complex, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium gordonae, Mycobacterium avium complex, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, and Mycobacterium chelonae - Mycobacterium abscessus complex. The results of LiPA were compared with the results obtained using traditional biochemical and molecular tests (DNA probe-based techniques, PCR restriction enzyme analysis of the 65 kDa heat-shock protein gene, and sequencing of the 16S rDNA). A total of 669 isolates, 642 of which were identified as Mycobacterium species and 27 as non- Mycobacterium species, were tested by LiPA. After analysis of 14 initially discordant results and exclusion of one isolate, concordant results were obtained for 636 of 641 Mycobacterium isolates (99.2% accuracy). All Mycobacterium species reacted with the MYC ( Mycobacterium species) probe (100% sensitivity), and all non- Mycobacterium species were identified as such (100% specificity).

Bacterial Typing Techniques↗

Use of specific rRNA oligonucleotide probes for microscopic detection of Mycobacterium avium complex organisms in tissue.

Members of the Mycobacterium avium complex (MAC) are important environmental pathogens that are implicated in several chronic, idiopathic diseases. Diagnosis of MAC-based diseases is compromised by the need to cultivate these fastidious and slowly growing organisms in order to identify which mycobacterial species are present. Detection is particularly difficult when MAC is intracellular or embedded within mammalian tissues. We report on the development of culture-independent, in situ hybridization (ISH) assays for the detection of MAC in culture, sputum, and tissue. This assay includes a highly reliable technique for the permeabilization of mycobacterial cells within culture and tissues. We describe a set of rRNA-based oligonucleotide probes that specifically detect either M. intracellulare, the two M. avium subspecies associated with human disease, or all members of MAC. The results call into question the validity of ISH results derived by the use of other gene loci, such as IS900.

Animals↗

Mycobacterium saskatchewanense sp. nov., a novel slowly growing scotochromogenic species from human clinical isolates related to Mycobacterium interjectum and Accuprobe-positive for Mycobacterium avium complex.

A pigmented, slowly growing Mycobacterium avium complex AccuProbe-positive organism was isolated from the sputum and pleural fluid of a 72-year-old female with bronchiectasis. The unusual morphology of the organism prompted further identification by 16S rRNA gene sequencing, revealing a perfect identity with previously uncharacterized strain Mycobacterium sp. MCRO 8 (GenBank accession no. X93034), with the closest established species by 16S rDNA analysis being Mycobacterium interjectum. HPLC of the organism corresponded to previously obtained patterns identified as M. interjectum-like and, upon sequence evaluation of a selection of strains with a similar profile, more were subsequently identified as MCRO 8. A total of 16 strains isolated from human respiratory samples were evaluated in the characterization of this novel species, for which the name Mycobacterium saskatchewanense sp. nov. is proposed. The type strain is strain 00-250(T) (=ATCC BAA-544(T)=DSM 44616(T)=CIP 108114(T)).

Bacterial Proteins↗

In-vitro activity of oxazolidinones against Mycobacterium avium complex.

Options for treating disseminated Mycobacterium avium complex (MAC) disease have improved. However, efficacy is not always certain, resistance is common and rapidly bactericidal agents would improve efficacy and prevent resistance. Certain oxazolidinones were tested against MAC strains and inhibited growth at expected serum concentrations or lower. Activity correlated with hydrophobicity and one agent was bactericidal at concentrations two to five times greater than the MIC.

Amikacin↗

Infection of "nonprofessional phagocytes" with Mycobacterium avium complex.

Organisms belonging to the Mycobacterium avium complex are the most common bacteria isolated from patients with AIDS. In these patients, M. avium is associated with disseminated disease, and bacteria are found within macrophages in the liver and spleen. To examine the potential of M. avium infection of a nonprofessional phagocyte, the murine fibroblast cell line (L929 cells) are infected with strain 101 (serotype 1) of M. avium. The duplication time for the intracellular bacteria was approximately 36 hr. Progressive intracellular growth ultimately resulted in the destruction of infected cells (after approximately 12 days in culture). Supernatant obtained from infected L929 cells contained interferon-beta (IFN beta) and granulocyte macrophage colony stimulating factor (GM-CSF) (50 +/- 12 ng/10(5) cells and 63 +/- 18 pg/10(5) cells, respectively, by 18 hr). IFN beta could be detected by 3 hr after infection, while GM-CSF was first detected by 6 hr. Release of IFN beta by infected L929 cells could subsequently stimulate NK cells, but not macrophages, to lyse infected L929 cells. These data using L929 cells suggest that M. avium may invade fibroblasts during the course of the infection and that fibroblast infection may trigger NK cell-mediated cytotoxicity against the infected fibroblasts.

Animals↗

Recombinant granulocyte-macrophage colony-stimulating factor activates human macrophages to inhibit growth or kill Mycobacterium avium complex.

Organisms belonging to the Mycobacterium avium complex (MAC) are associated with life-threatening bacteremia in patients with the acquired immunodeficiency syndrome (AIDS). As these organisms survive within macrophages, we examined the ability of recombinant human granulocyte-monocyte colony-stimulating factor (GM-CSF) to activate human monocyte-derived macrophages to inhibit the intracellular growth or kill the most mouse-virulent MAC strain in our collection that belongs to serotype 1. While unstimulated cells did not inhibit intracellular growth of MAC, macrophages activated by GM-CSF (10-10(4) U/ml) inhibited or killed up to 58 +/- 5% of the initial inoculum. This activation was dose-dependent, with maximal change occurring with a dose of 100 U/ml after 72 hr exposure. Inhibition or killing was demonstrated if GM-CSF was given both before or after establishment of infection. The combination of GM-CSF (10(2) U/ml) plus TNF (10(2) U/ml) augmented macrophage killing (range, 31 +/- 4%) compared with GM-CSF (10(2) U/ml) alone, but the combination of recombinant human interferon-gamma (IFN gamma) plus GM-CSF resulted in a significant decrease in intracellular inhibition of growth or killing (13.3 +/- 2%) compared with 57.7 +/- 5% obtained with GM-CSF alone. These results indicate that: 1) GM-CSF can activate macrophages to inhibit intracellular growth or kill MAC; 2) killing may be augmented by TNF; and 3) IFN gamma may impair GM-CSF-dependent macrophage activation.

Cell Survival↗

Two controlled trials of rifabutin prophylaxis against Mycobacterium avium complex infection in AIDS.

BACKGROUND: Disseminated Mycobacterium avium complex infection eventually develops in most patients with the acquired immunodeficiency syndrome (AIDS). This infection results in substantial morbidity and reduces survival by about six months. METHODS: We conducted two randomized, double-blind, multicenter trials of daily prophylactic treatment with either rifabutin (300 mg) or placebo. All the patients had AIDS and CD4 cell counts < or = 200 per cubic millimeter. The primary end point was M. avium complex bacteremia as assessed monthly by blood culture. The secondary end points were signs and symptoms associated with disseminated M. avium complex infection, adverse events, hospitalization, and survival. RESULTS: In the first trial, M. avium complex bacteremia developed in 51 of 298 patients (17 percent) assigned to placebo and 24 of 292 patients (8 percent) assigned to rifabutin (P < 0.001). In the second trial, bacteremia developed in 51 of 282 patients in the placebo group (18 percent) and 24 of 274 patients in the rifabutin group (9 percent) (P = 0.002). Rifabutin significantly delayed fatigue, fever, decline in the Karnofsky performance score (by > or = 20 percent), decline in the hemoglobin level (by more than 10 percent), elevation in alkaline phosphatase, and hospitalization. The incidence of adverse events was similar with rifabutin and placebo. Overall survival did not differ significantly between the two groups, although there were fewer deaths with rifabutin (33) than with placebo (47) during the double-blind phase (P = 0.086). The distribution of minimal inhibitory concentrations of rifabutin among the isolates of M. avium complex did not differ significantly between the treatment groups. CONCLUSIONS: Rifabutin, given prophylactically, reduces the frequency of disseminated M. avium complex infection in patients with AIDS and CD4 counts < or = 200 per cubic millimeter.

AIDS-Related Opportunistic Infections↗

Medical management of pulmonary disease caused by Mycobacterium avium complex.

The current medical therapy for Mycobacterium avium complex is controversial. American Thoracic Society recommendations advocate empiric therapy with drug susceptibility testing only for clarithromycin. At National Jewish, where we mainly see cases complicated by treatment failure and drug intolerance, we use in vitro susceptibility testing and therapeutic drug monitoring to optimize efficacy and reduce toxicity.

Anti-Bacterial Agents↗

The macrophage-induced gene mig as a marker for clinical pathogenicity and in vitro virulence of Mycobacterium avium complex strains.

The capacity of 20 Mycobacterium avium complex isolates to multiply intracellularly in human monocyte-derived macrophages was assessed and correlated to the clinical relevance of each isolate and its reactivity with several candidate genetic virulence markers. The strongest correlation with a virulence phenotype was found for a conserved coding sequence of the macrophage-induced gene mig identified by a specific mig restriction fragment length polymorphism type.

Biomarkers↗

Mycobacterium avium complex peritonitis in an AIDS patient.

Mycobacterium avium complex (MAC) frequently disseminates in AIDS patients, where the gastrointestinal tract is a major target organ. While ascites in AIDS patients is common, peritonitis secondary to MAC is rare. We describe the first case of MAC peritonitis in an AIDS patient without underlying cirrhosis, portal hypertension, chylous ascites or peritoneal dialysis. This case highlights the need to be aware of atypical presentations of MAC disease in AIDS patients with a history of disseminated MAC, even those who compliantly take highly active antiretroviral therapy.

AIDS-Related Opportunistic Infections↗

Activity of rifabutin, clarithromycin, ethambutol, sparfloxacin and amikacin, alone and in combination, against Mycobacterium avium complex in human macrophages.

Disseminated infection with Microbacterium avium complex (MAC) in patients with AIDS is currently treated with a combination of antimycobacterial agents in order to prevent the selection of resistant mutant strains. Although clinical and microbiological responses can generally be achieved within a few weeks, relapses are common and require modification of the combination regimen or identification of effective alternate therapies. In this study we investigated the activities of rifabutin 0.5 mg/L, sparfloxacin 1 mg/L, clarithromycin 4 mg/L, amikacin 16 mg/L and ethambutol 2 mg/L, alone and in combination, against nine strains of M. avium isolated from the blood of patients with AIDS in order to identify regimens with the greatest therapeutic potential. Macrophages derived from human monocytes were infected with M. avium and inoculated with a single drug or a combination of drugs; cfu counts were performed at 0, 4 and 7 days after infection. At day 4 and at day 7, the combination of rifabutin, clarithromycin, amikacin and sparfloxacin displayed the highest degree of activity. However, the activity did not differ significantly from that of the combination of rifabutin, clarithromycin and ethambutol. The results of this study confirm the activity of combinations including rifabutin and clarithromycin (+/- ethambutol) in human monocyte-derived macrophages and suggest potentially useful associations in incorporating sparfloxacin and amikacin.

Amikacin↗