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Illustrated in the image is the surface of a disk diffusion test including a 30 mg ceftazidime disk (left) and a combintation 30/10 mg ceftazidime/clavulanic acid disk (right). Observe in the photograph that the zone of inhibition around the the combination ceftazidime/clavulanic acid disk (right) is at least 5 mm larger than around the clavulanic acid disk (left). This observation that the presence of clavulanic acid, a beta-lactamase inhibitor, has resulted in such a large increase in the zone of inhibition indicates that an extended-spectrum beta-lactamase (ESBL)is being produced. Additionally, the Clinical and Laboratory Standards Institute (CLSI) Performance Standards for Antimicrobial Testing Standards, published January 2011, proposes in the M100-S21document, table 2A-S1, that cefotaxime (30 mg) and cefotazime-clavulanic acid (30/10 mg) testing per performed alone AND in combination with the ceftazidime and ceftazidime/clavulanic acid testing previously described. When an organism is producing an ESBL, the susceptibility to individual cephalosporins cannot be predicted, thus requiring that each drug must be tested individually.

Suppola JP. Kuikka A. Vaara M. Valtonen VV. Comparison of risk factors and outcome in patients with Enterococcus faecalis vs Enterococcus faecium bacteremia. Scandinavian Journal of Infectious Diseases. 30(2):153-7, 1998. The purpose of our study was to determine retrospectively the risk factors for the acquisition of Enterococcus faecalis vs E. faecium bacteremia, as well as the clinical outcomes of these patients. 62 patients with Enterococcus faecalis bacteremia were compared to 31 patients with E. faecium bacteremia. Haematologic malignancies, neutropenia, high-risk source and previous use of aminoglycosides, carbapenems, cephalosporins and clindamycin were significantly associated with E. faecium bacteremia. Instead, urinary catheterization was found to be related to Enterococcus faecalis bacteremia. The mortality rates within 7 d and 30 d were 13% and 27%, respectively, in patients with E. faecalis bacteremia and 6% and 29%, respectively, in patients with E. faecium bacteremia. There was no difference in mortality between E. faecalis and E. faecium bacteremia, nor was there a difference in seriousness of disease at the time of bacteremia. In the subgroups of patients with monomicrobial or clinically significant E. faecalis vs E. faecium bacteremia, the mortality rates were similar to the results of all subjects. Our results do not support the theory that E. faecium would be a more virulent organism than E. faecalis.

Lorimer JW. Eidus LB.: Invasive Clostridium septicum infection in association with colorectal carcinoma. Canadian Journal of Surgery. 37:245-9, 1994 The association between invasive Clostridium septicum infection and colorectal carcinoma is examined by the presentation of three cases and a review of the literature. In the first two cases the patients presented with nontraumatic metastatic clostridial gas gangrene. In the third case a patient with chemotherapy-induced myelosuppression from concomitant multiple myeloma had a necrotizing transmural infection of the right colon. The apparent portal of entry of Clostridium septicum was an occult carcinoma of the ascending colon. The increasing evidence for a strong link between this organism and some cases of neutropenic enterocolitis is reviewed.

Spencer RC.: Invasive streptococc European Journal of Clinical Microbiology & Infectious Diseases. 14 Suppl. 1:S26-32, 1995. Before the introduction of antibiotics, serious infections caused by Streptococcus pyogenes (Lancefield Group A streptococci) were common. Before World War II, this bacterium was responsible for as many as 50% of postpartum deaths and was the major cause of death in patients with burns. Also common were the sequelae of streptococcal infections-rheumatic fever and post-streptococcal glomerulonephritis. With the use of penicillin, however, Streptococcus pyogenes was believed to be virtually eliminated as a pathogen. The organism was consigned to the history books, but not for long. In the mid-1980s, focal resurgences of rheumatic fever began to be reported from different areas in the USA, such as Salt Lake City, Utah. In such communities, where increases in cases of rheumatic fever had been reported, the serotypes M-1, 3, 5, 6 and 18 were isolated which, on culture, produced characteristic mucoid colonies. At the same time, reports of increases in invasive streptococcal disease began to surface in both the US and Europe. Two syndromes were described; invasive streptococcal infection, occurring in previously healthy children and adults, commonly associated with septicaemia resulting from a deep focus of infection such as bone or lung; and streptococcal toxic shock syndrome, involving a cutaneous focus, accompanied by necrotizing or bullous soft tissue changes. Septicaemia is rare in streptococcal toxic shock syndrome, but the most characteristic feature is one of rapidly progressing multi-organ failure. A high proportion of the strains of Streptococcus pyogenes associated with this condition are serotype M-1, and fatality rates approaching 50% have been reported.

The stability of the CSF sample varies depending on the procedures ordered. Cell counts are ALWAYS STAT and should be performed within 30 - 60 minutes for best results. Samples should be left at room temperature for no longer than one hour and refrigerated following testing. Refrigeration is not recommended for culture specimens since fastidious organisms such as Haemophilus influenzae and Neisseria meningitidis may not survive the cold temperature.

The urine specimen should be freshly voided. Urine is an ideal medium for the proliferation of bacteria due to the large amount of urea present. These bacteria metabolize urea, producing ammonia that causes the urine pH to become more alkaline. If there is a delay before performance of the test, the sample should be refrigerated.

A molecular method may be the test method of choice for a variety of reasons, including the following:Organisms of interest, especially in microbiology or virology, may be extremely slow growing, fastidious, or extremely limited in quantityOther technologies lack the necessary sensitivity or specificityPatient population requirements

The mucicarmine staining procedure is very specific in its detection of mucins of epithelial origin and is used to identify adenocarcinomas. It is particularly useful in detecting adenocarcinomas originating in the gastrointestinal tract. This procedure can also successfully stain and detect the capsule of a fungal organism known as Cryptococcus neoformans . This fungus is usually identified in the lungs and in nervous tissues. C. neoformans is more likely to infect immunocompromised patients than persons with active immune systems.

Molecular diagnostics or molecular tests are assays that detect nucleic acids, DNA or RNA, in a sample. It is usually impossible to detect a whole strand of DNA or RNA; most often a specific region of DNA or RNA (called a target) that is unique to the organism, mutation, or disease is detected. Because these assays are detecting or quantifying biomolecules that are minute in size and concentration, special techniques are employed. Most assays include a component that is labeled and detection or measurement of the label is used to determine the presence or quantity of target in each sample.Two common techniques used in molecular testing are hybridization and amplification.

Biological agents are organisms or toxins that can kill or incapacitate people, live stock, and crops. The three basic groups of biological agents that would likely be used as weapons are bacteria, viruses, and toxins. Biological agents can be dispersed as aerosols or airborne particles.

Specialists in microbiology perform testing to diagnose and stop the spread of infectious organisms, including bacteria, viruses, and parasites. Specialists should be able to isolate and identify a wide variety of these organisms. Testing procedures include direction examination and antigen detection methods. Specialists in serology and immunology measure antibodies to infectious organisms. Specialists should be familiar with all serology techniques (except those specific to immunohematology). This specialty includes all lab procedures performed in the specialty of histocompatibility. Specialists in hematology must be able to identify and evaluate cells in blood and bone marrow and identify disorders of these cell. Specialists should be familiar with routine and special tests to determine the number, morphology, and function of cells in body fluid.

Human papilloma virus (HPV) is estimated to be the most common sexually transmitted infection in the United States. Digene's hybrid capture assay for HPV received approval by the Food and Drug Administration (FDA) in 2003. Only in recent years have other manufacturers, such as Third Wave Technologies, added this virus to their testing capabilities.Mycobacterium species represented another desirable target for molecular testing. Although some improvements in cultivation and staining techniques had been realized through the incorporation of broth media and fluorochrome staining, identification is still hampered by the growth rate of the organism. Gen-Probe first marketed probes that would allow identification of tuberculosis, M. avium-intracellulare, and M. gordonae in culture positive specimens. These probes greatly streamlined the workup of culture positive specimens.Of great interest to both clinicians and infection control practitioners, is the direct detection of Mycobacterium in clinical specimens. Gen-Probe received FDA approval for its AMPLIFIED MTDâ product for this specific application (in smear positive specimens) in 1995. This method employs isothermal transcription mediated amplification; the amplicons are detected using the same hybridization as the culture confirmation tests.

Even if an organism can survive various transport conditions and will be recovered in available culture systems with reliability, identification protocols may not provide clear cut differentiation, based on phenotypic tests and characteristics. Sequencing techniques are increasingly utilized for certain classes of organisms. Good examples are the aerobic, non-spore forming gram positive rods, including Nocardia and related genera, and mycology isolates. A more routinely encountered application occurs in virology. Molecular methods allow for efficient typing of herpes simplex (type I versus type II) and subtyping of specimens positive for influenza A.

In addition to instilling a consciousness about movement from one area to the next, other work skills are critical and key to obtaining accurate results:Aseptic techniquePipetting skillsAseptic technique Due to the sensitivity of molecular assays, there is little margin for error. Cross contamination from specimens with large numbers of organism/target nucleic acid can result from the slightest deviation in aseptic technique. The use of aerosol barrier pipette tips and positive displacement pipette devices go a long way in preventing aerosol contamination, but do not replace the attentiveness of the technologist during each transfer of specimen material. When working with specimens, only one specimen container should be opened at a time.Pipetting skills Although more and more assays are being introduced with pre-prepared master mixes, invariably some reagent preparation is required. This often entails the transfer of very small volumes, which leaves no margin of error during pipetting. Attentiveness to accuracy of pipetting is a must.

Another organism that has more recently become problematic is Clostridium difficile. Usually, normal gut flora resist overgrowth and colonization by this organism. However, antibiotic use that suppresses the normal gut flora, allows proliferation of C. difficile. The organism releases toxins that cause inflammation and damage to the mucosal lining of the colon, leading to severe diarrhea. An antibiotic-resistant strain has developed that can result in colitis, sepsis, and death. Elderly patients, patients with severe underlying illness, and patients undergoing immunosuppressive therapy are at higher risk of becoming infected since their immune response to the bacteria and its toxins is diminished.

S. aureus is the most pathogenic member of the genus Staphylococcus; it possesses several factors that contribute to its virulence: Structural components of its cell wall function as a protective barrier, aid in adherence to mucous membranes, and allow the organism to resist phagocytosis. The production of several different toxins Enterotoxins A, D, F (TSST1) Exfoliative toxin ( causing scalded skin syndrome Cytolytic toxins (causing cell & tissue damage). Production of enzymes Catalase – distinguishes staphylococci from streptococci Coagulase – distinguishes S. aureus from other staphylococci Hyaluronidase & lipase – aid in skin colonization/infection spread Beta-lactamase – breaks down the beta-lactam antibiotics, e.g., penicillins, cephalosporins, carbapenems and monobactams.

Surveillance is a critical component of any program for controlling multi-drug resistant organisms. Many institutions are using active surveillance cultures to identify patients who are colonized with a targeted MDRO. With respect to MRSA, an increasing number of hospitals are screening patients upon admission and on a periodic basis (usually weekly). The anterior nares is the primary site that is swabbed for screening.There are several selective and/or differential media that can be used for this purpose.Baird Parker Agar is a selective medium for the isolation of S. aureus; on this medium S. aureus produces black colonies with a clear halo.Mannitol Salt Agar is also a selective medium; S. aureus produces yellow colonies which contrast with the red color of the medium.Chromogenic agars have been developed for the isolation and presumptive identification of different species of bacteria and yeast. The media are formulated so that as different organisms utilize various substrates in the media, the organism of interest produce colonies with a unique color. Chromogenic agars specifically designed for the detection of MRSA are commercially available.In addition to culture methods, there are now commercially available, FDA approved methodologies for screening for MRSA by PCR. Although equipment and cost factors may not make these a viable option for every laboratory, they may offer greater sensitivity and improved turnaround times.

Clostridium are gram-positive or gram-variable, spore-forming, catalase-negative anaerobic bacilli. More than 100 species are currently recognized, though relatively few are encountered in properly collected clinical specimens from humans. There are three types of infection associated with Clostridium species: Non-invasive: Toxin-mediated Invasive: Progressive infection with tissue destruction Purulent disease: Closed space (e.g., in the peritoneal cavity) mixed infection with multiple organisms.Clostridium are well known as the agents of these classic toxin-mediated diseases : DISEASE TOXIN INVOLVED CAUSATIVE ORGANISM Tetanus or "lock jaw" Tetanospasmin Clostridium tetani Myonecrosis/Gas gangrene Exotoxins Clostridium perfringens Botulism (severe food poisoning) Botulin Clostridium botulinum

Clostridium difficile is the leading cause of hospital-acquired diarrhea in the United States, with the number of cases rising annually over the last three decades. This is largely due to the increased frequency of antibiotic usage, the development of better detection methods, and the fact that hospital environments are increasingly contaminated with spores of C. difficile. The definition of C. difficile diarrhea includes > 6 episodes of non-formed diarrheic stool per 24 hours, along with prior antibiotic treatment. At least three events must occur in the pathogenesis of C. difficile-associated diarrhea (CDAD): Alteration of the normal fecal flora Colonic colonization with toxigenic C. difficile Growth of the organism with elaboration of its toxins"Colonization resistance" is the term used to describe the mechanism by which indigenous flora control overgrowth of C. difficile. This resistance may be compromised by the use of antimicrobial compounds, underlying illness, or therapeutic procedures. Infection begins with the ingestion of either the organism itself or spores, usually via the fecal-oral route. Spores in particular are able to survive the acidity of the stomach and germinate in the colon to produce vegetative organisms. Toxinogenic strains subsequently produce Toxin A, Toxin B, and/or the Binary Toxin leading to colitis, pseudomembrane formation, and watery diarrhea. Significant complications of the clinical disease associated with infection are hypoalbuminemia, toxic megacolon (acute toxic colitis with dilatation of colon), and pseudomembranous colitis (PMC).

Cellular immunity includes delayed hypersentivity reactions, graft rejection, graft-versus-host reactions, defense against intracellular organisms, and probably defense against neoplasms.Cellular immunity is mediated by lymphocytes which we call T-cells.T-cells are so named because they are dependent on the thymus for their production and development.The majority of T-cells are long-lived with an average lifespan of 4.4 years, but it is known that some survive for as long as 20 years or more.T-cells are capable of leaving and re-entering the circulation many times during their long life.T and B cells cannot be differentiated when viewing blood films.They are identified through the use of immunologic cell markers.

As a health care worker, you come into contact with materials that may contain bloodborne pathogens. These are infectious organisms, usually viruses, that live in human blood and body fluids.The bloodborne pathogens that are of greatest concern to health care workers are:Hepatitis B virus (HBV) Human immunodeficiency virus (HIV) Hepatitis C virus (HCV)

As a healthcare worker, you come into contact with bloodborne pathogens. These are infectious organisms, usually viruses, which live in human blood and other potentially infectious body fluids.The most important ones are... Hepatitis B Virus (HBV) Human Immunodeficiency Virus (HIV)

This phlebotomist violated hospital procedures in several ways that could adversely impact patient care: Cleaning the site only with alcohol, not iodine, could result in a false-positive contaminated blood culture. This might result in the patient receiving unnecessary intravenous antibiotics, and could prolong the patients hospital stay unnecessarily. Drawing both cultures at the same time lessens the chance of recovering a bloodstream organism.Drawing both cultures from the same site might result in both of them being contaminated, making it very difficult for the physician to distinguish contamination from a "real" bloodstream infection.Relevant topics:Blood cultures: introduction, Avoid skin contamination, Blood culture site preparation 1, Blood culture site preparation 2

Blood is normally sterile. Any bacterial growth in the bloodstream is abnormal, and is an important cause of fever.Blood culture means the incubation of blood in appropriate media to allow growth and identification of bacteria or other organisms that may be present in a patient's bloodstream. Blood cultures are performed on febrile patients to identify and treat bloodborne organisms with the most appropriate antibiotic.

Clinical specimens where organism may be encountered: CSF Blood Stool (rare) Vesicle fluid, skin swab, or biopsy Gram stain morphology from clinical specimens: Large, gram-positive rods with square or concave ends in short chains Spores are usually NOT present Capsule may be viewed in smears from infected tissue, but this is NOT reliable Gram stain morphology from culture material: Large, gram-positive rods with square or concave ends, often in long chains (more than 2-4 cells) Cells easily decolorize as the culture ages Does NOT form capsules in culture Central to sub-terminal, oval spores, with NO significant swelling of the cell It must be noted that spore production increases with the age of the culture. Do NOT keep these cultures in the laboratory for longer than 24 hours for this reason!

Clinical specimens where organism may be encountered: Blood Biopsy, skin scraping, or swab Lymph node aspirate Respiratory secretions - oropharyngeal aspirate, sputum, or bronchial washingsGram stain morphology: Very tiny, gram-negative coccobacillus Pale or weak staining Due to the small size, often difficult to see individual cells

Francisella tularensis is a dangerous, highly infectious organism that can cause laboratory-acquired infections. It should NOT be manipulated on an open bench.Catalase: F. tularensis is weakly catalase positive. Catalase testing MUST be performed with extreme caution in a biosafety cabinet (BSC) due to the creation of aerosols. Oxidase: NegativeBeta-lactamase: PositiveUrease: NegativeXV factors: Not required for growthImportant note: F. tularensis is often incorrectly identified on automated identification systems. These systems may key out as Haemophilus influenzae or Actinobacillus species.

Burkholderia species is a dangerous and highly virulent organism that can cause laboratory-acquired infections. It should NOT be manipulated on an open bench.Catalase: Both organisms are catalase positive. Catalase testing MUST be performed with extreme caution in a biosafety cabinet (BSC) due to the creation of aerosols. Oxidase: B. mallei: Oxidase variable B. pseudomallei: Oxidase positiveIndole: Both organisms are indole negativeMotility: B. mallei: Non-motile B. pseudomallei: Motile

Brucella species: Brucella is distributed in nature worldwide and found in domesticated and wild animals, such as cattle, sheep, and pigs. Infection with Brucella species, known as brucellosis, is caused in humans by exposure to infected animal fluids or food products. This includes ingesting non-pasteurized dairy products, such as milk or cheese, inhaling aerosols, and skin contact with the fluids of infected animals. Brucellosis poses an increased risk of occupational exposure to laboratory, veterinary, and slaughterhouse workers. Brucella is the most commonly reported laboratory-associated bacterial infection.Burkholderia mallei and B. pseudomallei: Most Burkholderia are found in soil, but B. mallei is only found in mammals. B.mallei is the causative agent for Glanders which primarily affects animals such as donkeys, mules, and horses. Horses, the organism's natural host, are highly susceptible to infection. Human infection is rare and usually occurs in people working with infected animals or laboratory workers handling the organism. The organism is endemic in Africa, Asia, the Middle East, and Central and South America, and usually enters via the eyes, nose, mouth, abrasions or cuts in the skin, or through inhalation. B. pseudomallei is found in soil and water and can accidentally infect animals, plants, and rarely humans. It is the causative agent of melioidosis, which is endemic in areas of southeast Asia, Taiwan, and northern Australia. The organism generally enters through cuts in the skin, ingestion of contaminated water, or by inhalation of an aerosol.

The presence of many squamous epithelial cells (SQEs) also indicates a poorly collected urine specimen. If many SQEs are noted upon microscopic examination, the specimen should be recollected. The patient must be instructed how to collect a midstream, clean catch specimen. A Gram stain of a fresh, midstream urine sample would provide information that could help the physician decide whether to prescribe an antibiotic and the choice of antibiotic based on gram-reaction of the bacteria. Examine a Gram-stained slide made from a drop of uncentrifuged urine under oil immersion (1000X) magnification. If more than one bacterial organism is observed per oil immersion field, it can be determined that the quantity of bacteria is >105 colony forming units (CFU) per mL, and the patient probably has a urinary tract infection (UTI). The Gram stain reaction would also be important. Most bacteria that cause UTIs are gram-negative Enterobacteriaceae. A Gram stain report in this case would be "gram-negative bacilli consistent with quantity >105 CFU/mL."

When evaluating Gram stains of clinical samples, keep in mind the source of material from which the smear was made. Bacteria found in cerebrospinal fluid (CSF), blood, tissue and specimens from other sterile sites are always significant. Gram stains of body fluids that are normally sterile must be examined carefully. For every one organism per oil immersion field, there are about 105 organisms per mL present in the sample! Examining stained smears of CSF sediment may assist the clinician in establishing a presumptive diagnosis. The Gram stain result and the results of other special stains could also guide in the selection of culture media. If bacteria are observed in a CSF specimen, it is important to determine and report whether the bacteria are inside or outside of white blood cells (intracellular or extracellular). The quantity of organisms seen and the amount and type of host cells are also important to report. Bacteria observed in specimens from the throat, genital tract and other areas containing normal flora suggest infection only if their composition and type varies significantly from the norm.

The Gram stain reaction, shape, and arrangement of bacteria, and the presence or absence of intracellular organisms must be noted on the worksheet.Examples:Gram positive cocci in chains are present.Gram negative diplococci, intracellular, are present within white blood cells.Quantitate by approximating the average number of each cell type seen in 10 oil immersion fields, and record as:Many = More than 15/fieldModerate = 4-15/fieldFew = 1-3/fieldOccasional = 2-10/10 fieldsRare = 0-2/10 fields

Contamination of the staining solutions rarely occurs, but should be suspected when smears repeatedly contain the same organisms, and these organisms do not grow or are inconsistent with the clinical picture. Yeast and gram negative rods can occur as stain contaminants.

The ends of rod-shaped organisms may be rounded or tapered.

The following information is reported: Gram stain reaction ShapeThe cellular arrangement is not usually included in the report because it may vary, depending on the culture medium (liquid or solid) from which the organism was taken.

Polymerases are enzymes that synthesize DNA from an existing template. Polymerase requires a primer, nucleotides, and magnesium in order to function. In early PCR methods, the DNA polymerase was inactivated during the denaturation step. This required new polymerase to be added during each cycle of PCR. This problem was solved by the discovery of an enzyme termed Taq polymerase. Taq polymerase is isolated from the thermophilic organism Thermus aquaticus, a natural bacterium found in thermal springs. The Taq polymerase has optimal activity at 72°C but it can survive in temperatures up to 95°C. Today there are several other kinds of thermostable enzymes that are available for PCR, such as Pfu and Tli DNA polymerase. However, Taq is the DNA polymerase that is used most often in PCR procedures.

Bacteria may also be present, especially during a urinary tract infection. This view shows bacteria as solid gray rods or cocci. Since bacteria may also be a contaminant in specimens remaining at room temperature, or due to an unclean catch, caution must be observed in reporting bacteria. If 20 organisms per high power field (HPF) are seen, the bacteria are considered to be clinically significant.

Yersina entercolitica is most likely responsible for septic reactions in transfusions of Red Blood Cells. This organism is usually acquired by ingestion of contaminated food and causes mild symptoms of abdominal pain and diarrhea. Growth of Y. entercolitica is enhanced in iron-rich environments such as red blood cells. Other organisms reportedly found in Red Blood Cell units are Campylobacter species, Serratia species, Pseudomonas species, Enterobacter species, and Echerichia coli. These bacteria can produce endotoxins which cause a reaction in the patient. The majority of organisms associated with platelets transfusions are normal skin flora. Staphylococcus aureus, coagulase-negative staphylocci, aerobic and anerobic diptheroid bacilli, streptococci, and gram-positive bacilli are frequently isolated. Some transfused organisms have been implicated in a delayed post-transfusion illness. Pseudomonas aeruginosa and Burkholderia capacia have been isolated in cryoprecipitate and plasma. These organisms grow optimally at 30oC and have been found in water baths, accentuating the importance of overwrapping components that are thawed in a water bath. Rickettsia organisms are intracellular bacteria which are transmitted by ticks or insects. These bacteria are the causes of Rocky Mountain spotted fever, ehrlichiosis, and scrub typhus, and may be transmitted by transfusion. Similarly, the organism which causes Lyme disease may be transmitted as well. There have no reports of either of these organisms transmitted by transfusion.

TB infection is usually followed by an immune response and latency after exposure. In about 5-10% of cases, the latent period progresses to an active infection.Infection occurs when a susceptible person inhales droplet nuclei containing Mycobacterium tuberculosis and the organism reaches the alveoli of the lungs. The minimal infectious inoculum may be as low as one viable organism.About 2-12 weeks after infection, the immune system limits multiplication of additional bacteria and the immunological test becomes positive.Latent tuberculosis infection (LTBI) is the stage when the viable organism remains in the body; the individual has no symptoms and is noninfectious.Most persons infected with M. tuberculosis do not experience clinical illness and are noninfectious. About 5-10% of persons who are infected and are not treated will develop active TB during their lifetime. The risk for progression is highest during the first several years after infection.Most often, M. tuberculosis infects the lungs. However, it can infect almost any organ in the body, including bones and joints. Tuberculosis meningitis is a TB infection that occurs outside the lungs with devastating consequences, most often in young children and patients with AIDS.