Use of MALDI-TOF MS technology to evaluate adulteration of small ruminant milk with raw bovine milk.

Detection of adulteration of small ruminant milk is very important for health and commercial reasons. New analytical and cost-effective methods need to be developed to detect new adulteration practices. In this work, we aimed to explore the ability of the MALDI-TOF mass spectrometry to detect bovine milk in caprine and ovine milk using samples from 18 dairy farms. Different levels of adulteration (0.5, 1, 5, 10, 20, 40, 60, and 80%) were analyzed during the lactation period of goat and sheep (in May, from 60 to 90 d in milk, and in August, from 150 to 180 d in milk). Two different ranges of peptide-protein spectra (500-4,000 Da; 4-20 kDa) were used to establish a calibration model for predicting the concentration of adulterant using partial least squares and generalized linear model with lasso regularization. The low molecular weight part of the spectra together with the generalized linear model with lasso regularization regression model appeared to have greater potential for our aim of detection of adulteration of small ruminants' milk. The subsequent prediction model was able to predict the concentration of bovine milk in caprine milk with a root mean square error of 11.4 and 17.0% in ovine milk. The results offer compelling evidence that MALDI-TOF can detect the adulteration of small ruminants' milk. However, the method is severely limited by (1) the complexity of the milk proteome resulting from the adulteration technique, (2) the potential degradation of thermolabile proteins, and (3) the genetic variability of tested samples. Additionally, the root mean square error of prediction based only on one individual sample adulteration series can drop down to 6.34% for quantification of adulterated caprine milk and 6.28% for adulterated ovine milk for the full set of concentrations or down to 2.33 and 4.00%, respectively, if we restrict only to low concentrations of adulteration (0, 0.5, 1, 5, 10%).

Detection of bovine milk adulteration in caprine milk with N-acetyl carbohydrate biomarkers by using 1H nuclear magnetic resonance spectroscopy.

In a return to tradition, the popularity of caprine milk is on the rise. However, particularly in countries with developed dairy industries based on bovine milk, there is the risk of adulteration with bovine milk, which is a cheaper alternative. Thus, a rapid, robust, and simple method for the detection of bovine milk added to caprine milk is necessary, and 1H nuclear magnetic resonance spectroscopy appears to provide a solution. A matrix of 115 pure and artificially adulterated pasteurized milk samples was prepared and used to discover biomarkers of bovine milk that are independent of chemical and biological variation caused by factors such as genetics, diet, or seasonality. Principal component analysis and orthogonal projections to latent structures discriminant analysis of pure bovine milk and pure caprine milk revealed spectral features that were assigned to the resonances of 4 molecules. Of these, the peaks corresponding to protons in the N-acetylglucosamine and N-acetylgalactosamine acetyl moieties showed significant applicability for our method. Receiver operating characteristic curve analysis was used to evaluate the performance of the peak integrals as biomarkers of adulteration. This approach was able to distinguish caprine milk adulterated with 5% of bovine milk with 84.78% accuracy and with 10% of bovine milk an excellent 95.65% accuracy. This study demonstrates that N-acetyl carbohydrates could be used as biomarkers for the detection of bovine milk in caprine milk and could help in protecting caprine milk authenticity.

Polyphenols and health: Interactions between fibre, plant polyphenols and the gut microbiota.

A high-fibre diet and one rich in fruit and vegetables have long been associated with lower risk of chronic disease. There are several possible mechanisms underpinning these associations, but one likely important factor is the production of bioactive molecules from plant-based foods by the bacteria in the colon. This links to our growing understanding of the role of the gut microbiome in promoting health. Polyphenolic-rich plant foods have been associated with potential health effects in many studies, but the bioavailability of polyphenol compounds, as eaten, is often very low. Most of the ingested molecules enter the large intestine where they are catabolised to smaller phenolic acids that may be the key bioactive effectors. Dietary fibres, present in plant foods, are also fermented by the bacteria to short-chain fatty acids, compounds associated with several beneficial effects on cell turnover, metabolism and eating behaviour. Polyphenols and fibre are often eaten together, but there is a lack of research investigating the interaction between these two groups of key substrates for the colonic bacteria. In a project funded by the Biotechnology and Biological Sciences Research Council Diet and Health Research Industry Club, we are investigating whether combining different fibres and polyphenol sources can enhance the production of bioactive phenolic acids to promote health. This could lead to improved dietary recommendations and to new products with enhanced potential health-promoting actions.

Lactobacillus rodentium sp. nov., from the digestive tract of wild rodents.

Three strains of regular, long, Gram-stain-positive bacterial rods were isolated using TPY, M.R.S. and Rogosa agar under anaerobic conditions from the digestive tract of wild mice (Mus musculus). All 16S rRNA gene sequences of these isolates were most similar to sequences of Lactobacillus gasseri ATCC 33323T and Lactobacillus johnsonii ATCC 33200T (97.3% and 97.2% sequence similarities, respectively). The novel strains shared 99.2-99.6% 16S rRNA gene sequence similarities. Type strains of L. gasseri and L. johnsonii were also most related to the newly isolated strains according to rpoA (83.9-84.0% similarities), pheS (84.6-87.8%), atpA (86.2-87.7%), hsp60 (89.4-90.4%) and tuf (92.7-93.6%) gene sequence similarities. Phylogenetic studies based on 16S rRNA, hsp60, rpoA, atpA and pheS gene sequences, other genotypic and many phenotypic characteristics (results of API 50 CHL, Rapid ID 32A and API ZYM biochemical tests; cellular fatty acid profiles; cellular polar lipid profiles; end products of glucose fermentation) showed that these bacterial strains represent a novel species within the genus Lactobacillus. The name Lactobacillus rodentium sp. nov. is proposed to accommodate this group of new isolates. The type strain is MYMRS/TLU1T (=DSM 24759T=CCM 7945T).

Vagococcus entomophilus sp. nov., from the digestive tract of a wasp (Vespula vulgaris).

Three unknown Gram-stain-positive, catalase-negative, facultatively anaerobic and coccus-shaped strains of bacteria were isolated from the digestive tracts of wasps (Vespula vulgaris). Analysis of 16S rRNA gene sequences revealed that these strains had identical sequences and showed that Vagococcus salmoninarum, with 96.2% sequence similarity, was the closest phylogenetic neighbour. Further analyses based on hsp60 and pheS gene sequences of representatives of the family Enteroccocaceae and genotypic and phenotypic characterization using (GTG)5-PCR fingerprintings, EcoRI ribotyping, DNA G+C content, whole-cell protein profiling, cellular fatty acid profiles analysis and extensive biotyping confirmed that the investigated strains were representatives of a novel bacterial species within the genus Vagoccocus for which the name Vagoccocus entomophilus sp. nov. is proposed. The type strain is VOSTP2(T) ( = DSM 24756(T) = CCM 7946(T)).

Reclassification of Bifidobacterium stercoris Kim et al. 2010 as a later heterotypic synonym of Bifidobacterium adolescentis.

The taxonomic position of Bifidobacterium stercoris Eg1(T) ( = JCM 15918(T)) based on comparative 16S rRNA gene and hsp60 sequence analyses was found to be controversial, as the strain showed high similarity to the type strain of Bifidobacterium adolescentis, CCUG 18363(T). Therefore, the relationship between the two species was investigated by a taxonomic study that included, in addition to re-evaluation of the 16S rRNA gene sequence, determination of DNA-DNA binding and multilocus sequence analysis (MLSA) of housekeeping genes encoding the DNA-directed RNA polymerase B subunit (rpoC), putative xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (xfp), elongation factor EF-G (fusA), 50S ribosomal protein L2 (rplB) and DNA gyrase B subunit (gyrB). Comparative 16S rRNA gene sequence analysis showed relatively high similarity (98.9 %) between B. stercoris KCTC 5756(T) and B. adolescentis ATCC 15703(T). MLSA revealed close relatedness between B. stercoris KCTC 5756(T) and B. adolescentis CCUG 18363(T), with 99.3-100 % similarity between the rpoC, xfp, fusA, rplB and gyrB gene sequences. In addition, relatively high dnaJ1 gene sequence similarity of 97.7 % was found between the strains. Similar phenotypes and a high DNA-DNA binding value (78.9 %) confirmed that B. stercoris and B. adolescentis are synonymous. Based on these results, it is proposed that the species Bifidobacterium stercoris Kim et al. 2010 should be reclassified as a later heterotypic synonym of Bifidobacterium adolescentis Reuter 1963 (Approved Lists 1980).

Alloscardovia macacae sp. nov., isolated from the milk of a macaque (Macaca mulatta), emended description of the genus Alloscardovia and proposal of Alloscardovia criceti comb. nov.

A novel bacterial strain, designated M8(T), was isolated from milk of a female macaque bred in captivity. The strain was Gram-stain-positive, anaerobic, irregular coccoid-rod-shaped without catalase activity. Analysis of 16S rRNA gene sequence similarity revealed that the isolate was most closely related to Alloscardovia omnicolens CCUG 31649(T) (96.4%) and Metascardovia criceti OMB105(T) (96.6%). Sequences of hsp60, fusA, and xfp genes also confirmed that the strain was most closely related to the type strains of A. omnicolens and M. criceti. The isolate produced fructose-6-phosphate phosphoketolase which is in agreement with classification within the family Bifidobacteriaceae. The major fatty acids were C18 : 1ω9c (35.8%), C16 : 1 (6.2 %) and C14 : 0 (5.7 %). Polar lipid analysis revealed five different glycolipids, two unidentified phospholipids and diphosphatidylglycerol. The peptidoglycan was of the type A4α l-Lys-d-Asp with the presence of d(l)-alanine, d-glutamine, d-asparagine and l-lysine. The DNA G+C content of strain M8(T) was 50.1 mol%. On the basis of genetic, phylogenetic and phenotypic data, strain M8(T) represents a novel species of the genus Alloscardovia for which the name Alloscardovia macacae sp. nov. is proposed. The type strain is M8(T) ( = DSM 24762(T) = CCM 7944(T)). In addition, our results also revealed that Alloscardovia omnicolens DSM 21503(T) and Metascardovia criceti DSM 17774(T) do not belong to different genera within the family Bifidobacteriaceae. We therefore propose to reclassify Metascardovia criceti as Alloscardovia criceti comb. nov. An emended description of the genus Alloscardovia is also provided.

Pseudoscardovia suis gen. nov., sp. nov., a new member of the family Bifidobacteriaceae isolated from the digestive tract of wild pigs (Sus scrofa).

Seventeen fructose-6-phosphate phosphoketolase-positive bacterial strains were isolated from the digestive tract of wild pigs (Sus scrofa). Most of them were identified as Bifidobacterium boum according to sequences of 16S rRNA gene. Two strains isolated from the small intestine content had unusual morphology of cells in comparison with bifidobacteria. Cells growing in liquid anaerobic media were regular shaped rods arranged mostly in pairs. These isolates showed relatively low 16S rRNA gene sequence similarities (maximum identity of 94%) to members of the family Bifidobacteriaceae. Nevertheless, phylogenetic analyses of 16S rRNA, hsp60 and xfp gene sequences revealed that these strains are more related to recently described Neoscardovia, Aeriscardovia and other scardovial genera, than to Bifidobacterium species. Partial gene sequences of other phylogenetic markers showed low (65.8-89.5%) similarities to genome sequences of bifidobacteria and Gardnerella vaginalis. The major fatty acids detected in cells of the representative strain DPTE4(T) were C(16:0), C(18:1), C(14:0). The peptidoglycan type of the DPTE4(T) strain was A3ßl-Orn(l-Lys)-l-Ser(l-Ala)-l-Ala(2). Polar lipid analysis revealed two phosphoglycolipids and phospholipids, a glycolipid and diphosphatidylglycerol. The results of phylogenetic, genotypic and phenotypic analyses support the proposal of a novel taxa, Pseudoscardovia suis gen. nov., sp. nov. (type strain=DPTE4(T)=DSM 24744(T)=CCM 7942(T)).

Bifidobacterium actinocoloniiforme sp. nov. and Bifidobacterium bohemicum sp. nov., from the bumblebee digestive tract.

Our previous study, based primarily on PCR-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing, focused on the isolation of four bifidobacterial groups from the digestive tract of three bumblebee species. In that study, we proposed that these isolated groups potentially represented novel species of the family Bifidobacteriaceae. One of the four, Bifidobacterium bombi, has been described recently. Strains representing two of the other groups have been classified as members of the genus Bifidobacterium on the basis of positive results for fructose-6-phosphate phosphoketolase activity and analysis of partial 16S rRNA and heat-shock protein 60 (hsp60) gene sequences. Analysis of 16S rRNA gene sequence similarities revealed that the isolates of the first group were affiliated to Bifidobacterium asteroides YIT 11866(T), B. indicum JCM 1302(T) and B. coryneforme ATCC 25911(T) (96.2, 96.0 and 95.9 % sequence similarity, respectively), together with other bifidobacteria showing lower sequence similarity. Additional representatives of the second group were found to be affiliated to Bifidobacterium minimum YIT 4097(T) and B. coryneforme ATCC 25911(T) (96.0 and 96.3 % sequence similarity) and also to other bifidobacteria with lower sequence similarity. These results indicate that the isolates of the two groups belong to novel species within the genus Bifidobacterium. This observation was further substantiated by the results of partial sequencing of hsp60. On the basis of phylogenetic and phenotypic analyses and analysis of 16S rRNA and partial hsp60 gene sequences, we propose two novel species, Bifidobacterium actinocoloniiforme sp. nov. (type strain LISLUCIII-P2(T)  = DSM 22766(T)  = CCM 7728(T)) and Bifidobacterium bohemicum sp. nov. (type strain JEMLUCVIII-4(T)  = DSM 22767(T)  = CCM 7729(T)).

Bombiscardovia coagulans gen. nov., sp. nov., a new member of the family Bifidobacteriaceae isolated from the digestive tract of bumblebees.

One hundred and eighty-seven fructose-6-phosphate phosphoketolase positive strains were isolated from the digestive tract of three different bumblebee species. Analyses of the partial 16S rRNA gene sequences of the representative strains showed only 92.8% and 92.5% similarity to Bifidobacterium coryneforme YIT 4092(T) and Bifidobacterium indicum JCM 1302(T), 92.2% similarity to Alloscardovia omnicolens CCUG 18650 and slightly reduced similarity of 91% to other members of the family Bifidobacteriaceae. On the other hand, analyses of the partial heat-shock protein 60 (hsp60) gene sequence revealed that the proposed type strain BLAPIII-AGV(T) was affiliated only to the 60 kDa chaperonin sequence of uncultured bacteria from human vagina (79-80%) and the hsp60 gene sequence of A. omnicolens CCUG 31649(T) (75.5%). The peptidoglycan type was A4α with an l-Lys-d-Asp interpeptide bridge. The polar lipids contained diphosphatidylglycerol, an unknown phospholipid, six glycolipids and two phosphoglycolipids. The major fatty acids were C(18:1), C(20:0) and C(18:0). These and other analyses indicated that the isolates represented a new genus within the family Bifidobacteriaceae. This observation was further substantiated by determination of the DNA G+C contents (46.1-47.1 mol%). Affinity of the strains to some scardovial genera (Aeriscardovia, Alloscardovia and Metascardovia) was also confirmed by their ability to grow under aerobic conditions. Besides the above mentioned differences, Bombiscardovia coagulans was found to differ from all scardovial genera in the ability to grow at temperatures as low as 5°C, which was another major phenotypically different characteristic of this new member of the family Bifidobacteriaceae. Hence, on the basis of phylogenetic analyses using partial 16S rRNA and hsp60 gene sequence data, and the temperature related phenotypic difference, we propose a novel taxa, B. coagulans gen. nov., sp. nov. (type strain=BLAPIII-AGV(T)=DSM 22924(T)=ATCC BAA-1568(T)).

Bifidobacterium bombi sp. nov., from the bumblebee digestive tract.

Gram-positive-staining, anaerobic, non-spore-forming, lactate- and acetate-producing bacterial strains were isolated from the digestive tracts of different bumblebee species (Bombus lucorum, Bombus pascuorum and Bombus lapidarius). All of the isolates produced fructose-6-phosphate phosphoketolase activity. A representative strain, BluCI/TPT, was characterized further. Cells of strain BluCI/TPT showed occasional bifurcation and irregular constrictions. The bacterium utilized a wide range of carbohydrates. Glucose was fermented to acetate and lactate. The DNA base composition was 47.2 mol% G+C. Complete 16S rRNA and partial hsp60 gene sequences were obtained and phylogenetic relationships were determined. Strain BluCI/TPT and related isolates were located in the actinobacterial cluster and were closely related to the genera Bifidobacterium, Scardovia, Aeriscardovia and Parascardovia. The results presented support the proposal of a novel species to accommodate strain BluCI/TPT, with the name Bifidobacterium bombi sp. nov.; the type strain is BluCI/TPT (=DSM 19703T=ATCC BAA-1567T).

[The importance of pneumococcal vaccination - a clinician's view]. / Význam ockování proti pneumokokum - pohled klinického pracovníka.

Reliable epidemiological surveillance of pneumococcal diseases is crucial for rational decisions concerning the introduction of vaccination strategies. In countries with such close surveillance (e.g. in the USA or Belgium), the acquired epidemiological data warranted universal vaccination. Pneumococci cause short-time asymptomatic colonization of the human nasopharynx. If defense mechanisms fail, pneumococci may penetrate to previously sterile areas and cause local infections in the upper airways. In case they reach the bloodstream, invasive infections occur that threaten health or even lives of both children and adults. Therapeutic options are limited due to increasing resistance of pneumococci to antibiotics. Possible prevention of particularly invasive infections is vaccination of adults with a 23-valent polysaccharide vaccine and children over two months of age with a polyvalent conjugate vaccine. Therefore, the WHO recommends that the pneumococcal conjugate vaccine is included in national vaccination programmes as soon as possible.

Comparison of chemical composition and antibacterial activity of Nigella sativa seed essential oils obtained by different extraction methods.

Nigella sativa L. seed essential oils obtained by hydrodistillation (HD), dry steam distillation (SD), steam distillation of crude oils obtained by solvent extraction (SE-SD), and supercritical fluid extraction (SFE-SD) were tested for their antibacterial activities, using the broth microdilution method and subsequently analyzed by gas chromatography and gas chromatography-mass spectrometry. The results showed that the essential oils tested differed markedly in their chemical compositions and antimicrobial activities. The oils obtained by HD and SD were dominated by p-cymene, whereas the major constituent identified in both volatile fractions obtained by SD of extracted oils was thymoquinone (ranging between 0.36 and 0.38 g/ml, whereas in oils obtained by HD and SD, it constituted only 0.03 and 0.05 g/ml, respectively). Both oils distilled directly from seeds showed lower antimicrobial activity (MICs > or = 256 and 32 microg/ml for HD and SD, respectively) than those obtained by SE-SD and SFE-SD (MICs > or = 4 microg/ml). All oil samples were significantly more active against gram-positive than against gram-negative bacteria. Thymoquinone exhibited potent growth-inhibiting activity against gram-positive bacteria, with MICs ranging from 8 to 64 microg/ml.

[Primary retrofascial abscess in the iliopsoas muscle caused by salmonella enteritidis.]. / Primární retrofasciální absces iliopsoatického svalu zpusobený Salmonelou enteritidis.

This case report and review of literature demonstrates a rare entity of a primary retrofascial abscess in the iliopsoas muscle caused by Salmonella enteritidis, that progressed into the iliac fossa. It is of importance to the orthopedic surgeon in his differential diagnosis of hip pain and vertebrogenic disorders. It can also resemble other infectious diseases or tumors as it occurs mainly in young or imunosuppressed individuals. The primary and secondary etiology of this disease and it's different names are discussed together with the underlying bacteria. Clinical, laboratory and radiological findings are compared as well as the outcome of different types of treatment. When clinically suspecting this disease it is useful to confirm it with sonography and then perform CT scans for accurate planing of surgery. n the initial phase a CT guided drainage together with antibiotics and exceptionally only conservative treatment can cure the patient. In delayed cases it is necessary to preform an open drainage and cases with fatal outcomes have also been reported mainly in secondary abscesses. If early diagnosed and properly treated, this disease can be easily managed, therefore we have to keep it in mind in our differential diagnosis of indicated cases. Key words: primary pyogenic retrofascial abscess of the iliopsoas muscle, iliac fossa abscess, nontuberculous psoas abscess, psoitis, Salmonella enteritidis, clinical diagnosis, radiological diagnosis, surgical treatment, antibiotic treatment, ethiology.

Ceftriaxone in the treatment of Lyme neuroborreliosis.

In an open non-comparative clinical trial with the aim of evaluating the clinical efficacy and safety of a 14 day course of 2 g ceftriaxone once daily, 46 patients with neuroborreliosis were entered at the Infectious Diseases Teaching Hospital in Prague 8. In 39 patients the diagnosis was early Lyme neuroborreliosis. Seven patients suffered from late stage disease. Clinical results were 30% of patients cured at the end of treatment and 85% after 9 months in early stage disease. In late stage disease two patients out of seven were cured and four had improved after 12 months. One patient died because of cardiac infarction. In no patient had treatment to be discontinued because of adverse reactions to antibiotics.

Long-term results in patients with Lyme arthritis following treatment with ceftriaxone.

A total of 35 patients with late stage Lyme borreliosis with involvement of the joints was followed up until 3 years after a 14 day course of 2 g ceftriaxone once daily i.v. Diagnosis was confirmed by indirect and direct microbiological methods as well as clinical signs and symptoms. Long term clinical results in 26 patients at 36 months were complete response or marked improvement in 19, relapse in six and new manifestations in four of the cases, respectively. Possible mechanisms for non-responding to therapy are discussed. Therapy was well tolerated; in no case discontinuation of treatment was necessary due to adverse drug reactions. The treatment results in this group of 35 patients with Lyme arthritis are considered successful. The data obtained are consistent with expectations based on the published experiences with ceftriaxone in this indication.

Predictors of survival in patients with AIDS and disseminated Mycobacterium avium complex disease.

Patients with AIDS and disseminated Mycobacterium avium complex disease (DMAC), as defined by the presence of a positive blood culture for MAC, were studied retrospectively to define the natural history of DMAC. All patients had fevers, severe anemia (hematocrit < 26%), or both. Eighty-seven (76%) had signs, symptoms, or laboratory findings related to the gastrointestinal tract, but no distinct syndrome was identified. Sixty-nine patients received antimycobacterial therapy; assignment to therapy was not randomized. In a proportional hazards analysis, shorter survival was associated with higher initial level of mycobacteremia (relative risk [RR], 1.86; 95% confidence interval [CI], 1.49-2.31; P < .001), while administration of antimycobacterial chemotherapy (RR, 0.42; 95% CI, 0.26-0.70; P < .001) and antiretroviral therapy (RR, 0.40; 95% CI, 0.22-0.73; P < .01) had protective effects. Thus, the initial level of mycobacteremia of patients with DMAC may have prognostic value, and administration of antimycobacterial and antiretroviral agents may be associated with prolonged survival.

Low serum levels of oral antimycobacterial agents in patients with disseminated Mycobacterium avium complex disease.

Twenty-seven human immunodeficiency virus-infected patients with disseminated Mycobacterium avium complex disease who were treated with oral antimycobacterial agents (clofazimine, ciprofloxacin, ethambutol, and rifampin) were studied to evaluate the usefulness of monitoring serum drug concentrations and testing in vitro susceptibility of M. avium complex (MAC) isolates. Twenty patients tolerated treatment with three or four antimycobacterial agents for at least 8 weeks; mycobacteremia was eradicated in 7 (35%). The in vitro susceptibilities of MAC isolates to antimycobacterial agents were similar for these 7 and for the 13 who did not respond to antimycobacterial treatment. Serum drug levels were below the expected range in 6 of the 7 whose mycobacteremia was cleared and in 9 of the 13 nonresponders (P = .41). These low serum concentrations of antimycobacterial drugs may be due to impaired drug absorption in patients with AIDS and disseminated MAC disease.

A prospective evaluation of Mycobacterium avium complex colonization of the respiratory and gastrointestinal tracts of persons with human immunodeficiency virus infection.

To describe the natural history of Mycobacterium avium complex (MAC) in the respiratory or gastrointestinal tract of persons with human immunodeficiency virus (HIV) infection, 67 HIV-infected patients with CD4+ cell counts < 200/mm3 and initial negative MAC blood cultures were followed prospectively. Patients were screened every 3 months with cultures and smears of sputum, rectal swab, and blood for mycobacteria. Fourteen patients (20.9%) developed positive blood cultures for MAC (23.4%/year). Sputum cultures revealed MAC in 3 (21%) of the 14 patients at 1, 2, and 8 months before dissemination; no smears were positive. No rectal swab cultures or smears were positive before dissemination. Colonization of the respiratory and gastrointestinal tracts in persons with HIV infection and < 200/mm3 CD4+ cells is infrequently detected with currently available techniques. Screening cultures and smears of sputum and stool do not appear to be sensitive methods for detection of early MAC infection.