Articles on Medical Diseases and Conditions

Author: admin

  • Gram Stain

    Gram staining provides a presumptive diagnosis and some indication of the organism involved without waiting for culture results. On occasion, Gram stain may reveal organisms that (for technical reasons) do not grow when cultured. Best information comes from areas that are normally sterile. Gram staining is considered a routine procedure for CSF in possible meningitis, for urethral smears in possible venereal disease, and for material from abscesses or effusions. (This is especially true when anaerobes may be present. These may fail to grow, since “anaerobic” culture is often suboptimal, but the organisms might be seen on a Gram stain.) In certain other types of specimens, such as urine or stool, a Gram stain need not be done routinely but should be performed in special circumstances, such as on stool specimens when pseudomembranous enterocolitis is suspected or on urine specimens if quantitative culture is not available.

    The Gram stain is controversial in several other areas. The most important example is sputum. One can find opinions in the literature on the usefulness of a sputum Gram stain ranging from “essential” to “worthless and misleading.” One major problem is contamination by normal organisms from the mouth and nasopharynx. Certain pathogens, such as pneumococci or S. aureus, may be located in the upper respiratory tract of many clinically normal persons, and these may gain entry into the sputum. Similarly, upper respiratory tract colonization by enteric gramegative organisms is relatively frequent in alcoholics and in patients who have been hospitalized for more than a few days. Another difficulty is occasional discovery of opportunist organisms in the lower respiratory tract, organisms not normally present but not apparently causing disease. Another problem is the morphologic similarity of pneumococci and streptococci, among others. Finally, and most important, the sputum sample may, in fact, be only saliva.

    There are certain maneuvers that may help to circumvent some of these drawbacks. The number of organisms may provide a clue to their significance. Marked predominance or heavy growth of one organism suggests true abnormality. Many polymorphonuclear leukocytes is some evidence in favor of acute infection. Significant quantities of squamous epithelial cells (>10 per low-power field) usually means oral contamination. The patient should be instructed how to produce a deep cough and, if possible, should be observed while he or she produces the specimen. In some cases aerosol therapy may be helpful to induce sputum.

  • Sputum Culture

    The usefulness of sputum culture is controversial. This method of diagnosis has evoked the same spectrum of emotions and suffers from most of the same potential drawbacks as Gram stain of sputum. Various studies have demonstrated that either sputum or bronchoscopic specimens are frequently contaminated by upper respiratory tract bacteria. Some of the contaminants, such as Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, enteric gramegative bacteria, and Candida organisms, are potential lower respiratory tract pathogens. In addition, bronchoscopy may introduce local anesthetic into the specimen. Transtracheal aspiration (insertion of a needle into the trachea) or direct needle aspiration of the lung has been shown to produce relatively uncontaminated specimens. However, these techniques have potential complications. Although there is general agreement on the possibility of contamination, there is difference of opinion in the literature on the possibility that sputum culture may sometimes fail to detect the bacteria responsible for pneumonia. The importance of a specimen from the lower respiratory tract rather than the mouth or nasopharynx must be reemphasized, especially in seriously ill, uncooperative, or mentally impaired patients. As mentioned previously, a “pure culture” or marked predominance of one organism enhances suspicion of pathogenicity.

    Several studies have found that the number of squamous epithelial cells in sputum provides a useful index for degree of oropharyngeal contamination; the greater the number of squamous epithelial cells per low-power field (10 x microscope objective), the more likely to have significant degrees of contamination. There is some disagreement in the literature between 10 or 25 squamous epithelial cells per low-power field as the criterion cutoff number. Significant contamination strongly suggests that a new specimen should be collected with special attention given to obtaining true lower respiratory tract material with a minimum of oral contamination. Some investigators also quantitate the number of segmented neutrophils (WBCs) per low-power field; more than 25 suggests acute inflammation (however, this alone does not differentiate upper from lower respiratory tract origin).

  • Urine Culture

    Contamination of urine specimens by vaginal or labial bacteria is a serious problem. The most reliable way to obtain the specimen, especially in young children, is by suprapubic puncture and aspiration of the bladder with a needle and syringe. However, this technique has never become popular. Catheterization is another way to solve the difficulty, but approximately 2% of patients are reported to develop urinary tract infection following a single catheterization. A midstream “clean-catch” voided specimen is the third best procedure. The urinary opening is cleansed. The labia are held apart in the female and the foreskin pulled back in the male. The first portion of urine is allowed to pass uncollected, then the sterile container is quickly introduced into the urine stream to catch the specimen.

    Quantitative urine cultures have now replaced simple cultures, since it has been generally accepted that a titer of 100,000 (105) or more organisms/ml has excellent correlation with clinical infection, whereas a titer of less than 1,000 (103) organisms/ ml is usually not significant. However, there are certain important reservations and exceptions to this commonly accepted statement. The original studies that established this concept were performed on nonsymptomatic women using clean-catch urine collection. In addition, it was found that a single urine specimen collected this way that contained more than 100,000 organisms/ml had only an 80% chance of representing true infection (i.e., there was a 20% chance that the specimen was contaminated). Two consecutive urine specimens containing more than 100,000 organisms/ml has a 95% chance of indicating true infection. If the patient is receiving antibiotics or if the specimen is obtained by catheterization, many authorities accept a colony count of 1,000 or more organisms/ml as significant. If the specimen is obtained by suprapubic puncture or by cystoscopy, any growth at all is considered significant. If the patient has symptoms of urinary tract infection (fever, chills, flank pain, and pyuria), many authorities are willing to accept a clean-catch specimen colony count of 1,000 or more organisms/ml as significant even if the patient is not taking antibiotics. Not all patients with symptoms of urinary tract infection have colony counts over 1,000 organisms, because some females who have dysuria and frequency have urethritis (acute urethral syndrome, discussed earlier) rather than cystitis. About 20%-30% of these cases of urethritis are due to Chlamydia trachomatis, which does not grow on standard urine culture media. There is also the possibility that some cases could be chlamydial in origin but that the urine culture becomes contaminated with other bacteria, which leads to a false impression of cystitis. Finally, in some cases of pyelonephritis there are few or intermittent symptoms, and the urine cultures may be consistently negative, intermittently positive, or contain relatively low numbers of organisms. If there is a strong suspicion of pyelonephritis, repeat cultures may be necessary if the initial culture is negative or equivocal.

    Use of the quantitative (titer) technique with a cutoff level of 100,000 organisms/ml is supposed to compensate for small degrees of unavoidable contamination, but contamination can easily be severe enough to give a positive result. Various studies using midstream specimens have shown a surprisingly high rate of contamination in the female. In my experience, a microscopic examination of centrifuged specimen sediment in which more than 10 squamous epithelial cells per low-power field (10 x ocular, 10 x objective) are demonstrated suggests possible contamination. Three or more species of bacteria isolated from the same urine specimen also suggests contamination if the patient is not on long-term indwelling catheter drainage. All available information emphasizes that good technique in collecting the specimen is essential. Also essential is getting the specimen to the laboratory as soon as possible after collection. Urine is an excellent culture medium, and specimens that stand for more than 1 hour at room temperature permit bacterial incubation and proliferation to the point that quantitative counts are not reliable. If delivery to the laboratory must be delayed, the specimen should be refrigerated. The specimen can be preserved up to 12 hours in a refrigerator (4°C).

    Quantitative urine culture has two other drawbacks. Culture involves trained technical personnel and relatively expensive media, thus curtailing use for mass screening to detect urinary tract infection. Culture takes 24 hours to determine bacterial quantity and another 24-48 hours to identify the organism, thus delaying treatment in suspected infection.

    Screening tests for bacteriuria

    Because of the delay inherent in culture methods, several screening tests for rapid detection of significant degrees of bacteriuria have been introduced. If organisms are seen on Gram stain of uncentrifuged urine, reports indicate about 93% (range, 88%-95%) probability of a positive (100,000 organisms/ml) quantitative culture. The same is said to be true of direct examination of unstained centrifuged urine sediment. Gram-stained centrifuged sediment apparently gives too many false positive results. Gram-stained uncentrifuged urine and unstained urine sediment examination are said to give relatively few false positive results. Microscopic examination of centrifuged urine sediment to detect pyuria (increased number of WBCs) is widely used to screen for urinary tract infection. However, pyuria is found in only about 70%-80% (range, 13%-93%) of patients with positive urine cultures, and about 30% (range, 2%-40%) of patients with pyuria do not have a positive urine culture. For example, in one group of infants with culture-proven urinary tract infection, 42% had normal urine sediment and no bacteria seen on Gram-stained smears of noncentrifuged urine. Leukocyte esterase dipsticks have been used as a substitute for microscopic examination. They are positive in about 80%-90% (range, 72%-98%) of patients with a positive culture. Some urine test dipsticks have both the leukocyte esterase and a biochemical nitrite test; if results of both are positive, this detects 85%-90% (range, 78%-100%) of positive results from culture. Several chemical methods have been advocated. The most successful include the triphenyl tetrazolium chloride (TTC) test, the Greiss nitrate test (previously mentioned), and the catalase test. Best results have been reported with the TTC test. Nevertheless, reports vary as to its accuracy, with a range of 70%-90% correlation with quantitative culture, including both false positive and false negative results. The Greiss nitrate test detects about 55% (range, 35%-69%) of cases with a positive culture. The catalase test is even less reliable.

    Finally, there are several other bacteriuria screening systems now available, and more keep appearing. The Marion Laboratories Bac-T-Screen filters the urine, trapping any bacteria on the filter, which is then stained to reveal presence of bacteria. Sensitivity (compared to culture) is reported to be about 93% (range, 88%-99%). However, false positive results are about 29% (range, 16%-55%). Two companies market tests using firefly luciferase (bioluminescence) that reacts with bacterial ATP. Three companies market automated screening instruments, two detecting turbidity produced by bacterial growth (Autobac and MS-2 systems) and one (AMS systems) scanning a multiwell card containing various test media. Results from all of these systems are similar to those of the Bac-T-Screen. The advantages of these systems are that negative test results suggest that culture would not be necessary. Results are available the same day. Drawbacks are that positive test results by any of these methods must be followed by quantitative culture (except possibly the AMS system). False positive test results are relatively frequent, and negative test results do not completely rule out urinary tract infection (especially since the sensitivity figures quoted earlier are for 100,000 organisms/ml, and detection rates become less if bacterial numbers are less).

    Drawbacks of urine quantitative culture

    In summary, although quantitative culture with a cutoff titer value of 100,000 organisms/ml is widely considered the most reliable index of urinary tract infection, there are several major limitations:

    1. Using a titer of 100,000 organisms/ml as the cutoff point for a positive culture decision eliminates specimens with low-grade contamination but does not identify those with greater degrees of contamination.
    2. The cutoff point of 100,000 organisms/ml does not apply to catheterized or suprapubic aspiration specimens and may be misleading if used to interpret them.
    3. There are circumstances in which fewer than 100,000 organisms/ml may be significant in clean-catch specimens.
    4. A positive culture does not reveal which area of the urinary tract is involved.
    5. Tuberculosis of the urinary tract, anaerobic infections, and chlamydial infections of the urethra give negative culture results on ordinary culture media.
    6. Bacteremia from many causes, even if transient, involves organisms that are filtered by the kidney and may produce a temporarily positive quantitative culture result.
    7. Some cases of urinary tract infection may give negative cultures at various times. In several studies, a sizable minority of patients required repeated cultures before one became positive. This may be due to the location of the infection in the kidney and to its degree of activity.

    Therefore, two cautions are required when one is interpreting a urine culture report. If the results suggest that urinary tract infection is present, the physician must be certain that the specimen was properly collected, especially in the female. If the result is negative, this does not rule out chronic pyelonephritis. The problem of pyelonephritis and bacteriuria is also discussed in Chapter 12.

    Kits with agar-coated slides that not only yield quantitative urine culture results but also act as a culture medium for the organisms are now available. These are reported to have 90%-98% accuracy in various university hospital laboratories when compared with standard quantitative culture methods. The average is about 95% reliability. There is some question whether office laboratories can approach this figure and what provision would be made to identify the organisms.

    It should be mentioned that a few studies have reported little or no difference in contamination rates between clean-catch cultures and no-precaution cultures.

  • Obtaining a Specimen for Culture

    After material has been taken for culture, three steps should be followed. First, the specimen must be taken to the laboratory as soon as possible, since many organisms die on prolonged exposure to air or drying. This is especially true for swab preparations. Swab kits are available that contain a carrier medium into which the specimen is placed. This is a great help in preserving most bacteria, but the medium is not ideal for all organisms. For example, gonococci or anaerobes must have special transport systems. Anaerobic specimens require special precautions when the specimen is obtained and during transport to the laboratory. Second, the source of the culture should be written on the request sheet. This tells the laboratory what normal flora organisms are to be expected and provides some information on the pathogens that should be looked for and thus what media should be used. Finally, if a specific organism is suspected, this information should also be written on the request, so that if special culture methods are required, the requisite techniques will be anticipated and used.

  • General Isolation and Identification Techniques

    It is useful to know certain technical information involved with isolation and identification of bacteria. This information may improve physician understanding of the microbiology laboratory, to the mutual benefit of physician and laboratory.

    If a culture is ordered, it usually takes at least 48 hours, and often longer, for definitive diagnosis—1 day to culture the organism and 1 day to identify it. Sometimes an additional day must be used to isolate the organism if it is present in a mixed culture. One additional day may be needed for antibiotic sensitivities. A technologist uses knowledge of the site and source of culture material in deciding what media or techniques (e.g., anaerobic conditions) to use for isolation. Some organisms are normal inhabitants of certain body areas but pathogens in other areas, so an experienced technologist knows to some extent what to subculture from a mixture of organisms growing in a specific location and also what special media to use for the pathogens usual in that anatomical location. This knowledge can easily save 1 day’s time. Information that a certain specific organism is suspected may save even more time by permitting original inoculation of the culture material into special test media. Even if definitive isolation is not yet accomplished, the technologist can often provide useful information or even a presumptive diagnosis. For example, among the enteric gramegative rods, Escherichia coli, Enterobacter, and Klebsiella ferment lactose, whereas most of the other pathogens do not. Therefore, a lactose fermenter cannot be Salmonella or Shigella. Pseudomonas, in addition, does not ferment glucose, whereas most of the other pathogens do. Some organisms have a fairly characteristic appearance on isolation media. For example, it may be possible to make a rapid presumptive diagnosis for some of the gram-positive aerobic bacteria based on their appearance on original blood agar culture plates combined with their Gram stain morphology.

  • Bacteria Associated with Contamination of Specimens

    Certain bacteria that ordinarily are nonpathogenic (such as normal inhabitants of certain areas) frequently appear in cultures and are traditionally considered contaminants, presumably introduced by faulty culture technique. The major species are Staphylococcus epidermidis and the diphtheroids. Occasionally there are others, such as Bacillus subtilis. However, under certain circumstances, especially when the patient is immunocompromised, these organisms may produce disease. Three or more species of bacteria appearing in culture from ordinarily sterile areas (e.g., blood, spinal fluid, body cavity fluid, urine without a long-term indwelling catheter) suggest contamination. However, true polymicrobial infections may occur.

    Bacterial species omitted. Discussion of bacterial infections in this chapter has been limited to the common organisms in clinical practice and a few that enter the differential diagnosis of certain common situations, such as hepatitis or fever of unknown origin. Many others have been omitted, such as Haemophilus ducreyi, which produces the venereal disease chancroid, and Borrelia spirilis, which causes relapsing fever. In general, most are diagnosed through culture of appropriate specimens. The reader is referred to standard textbooks on microbiology for additional information on these organisms.

  • Miscellaneous Bacteria

    Whipple’s disease. This is a rare systemic illness that involves the small intestine mucosa but also may have low-grade fever, arthralgias, lymphadenopathy, and CNS symptoms (confusion, loss of memory, vision abnormalities, and symptoms referable to involvement of one or more cranial nerves). The GI symptoms are primarily those of malabsorption similar to sprue; with steatorrhea, weight loss, abdominal pain, and hypoalbuminemia (due to loss into the GI tract). Diagnosis currently involves biopsy of the duodenal mucosa, which contains many PAS stain-positive macrophages in the mucosa. A bacillus-type infection has been postulated on the basis of electron micrograph findings, but no organism has been cultured that could be proven to be the responsible agent. Recently, two groups of investigators using nucleic acid probe methods have identified a bacillus that appears to be a gram-positive member of the actinomyces family, tentatively named Tropheryma whippelii. Culture has not yet been possible and this discovery still must be definitively established.

  • Other Bacteria of Medical Importance

    Corynebacteria

    These organisms are gram-positive aerobic rods of varying length, frequently arranged on smear in sheafs (palisades) or Chinese letter-type configurations. The most important member of this genus is Corynebacterium diphtheriae, which causes diphtheria. Infection usually becomes established in the pharynx. Corynebacterium diphtheriae produces a toxin that affects the heart and peripheral nerves. There is also necrosis of the infected epithelium, with formation of a pseudomembrane of fibrin, necrotic cells, and neutrophils. The disease is now uncommon, but cases appear from time to time, some of them fatal. Contrary to usual belief, the laboratory cannot make the diagnosis from Gram-stained smears. Heat-fixed smears stained with methylene blue are better but, although helpful in demonstrating organisms resembling diphtheria, they still are not reliable enough for definite morphologic diagnosis. Nonpathogenic diphtheroids are normal nasopharyngeal inhabitants and may look very much like C. diphtheriae. To be certain, one must culture the organisms on special Loeffler’s media and do virulence (toxin production) studies. Nevertheless, direct Gram-stained and methylene blue smears are of value, since other causes of pharyngeal inflammation and pseudomembrane formation, such as fungus and Vincent’s angina, can be demonstrated. Therefore, two or three pharyngeal swabs should be obtained, if possible, for smears and for cultures. About 20% of cases will be missed if specimens for culture are not obtained from the nasopharynx as well as the pharynx.

    Certain nondiphtheritic species of Corynebacterium are called diphtheroids. These are normal skin and nasopharyngeal inhabitants and appear rather frequently in cultures as contaminants. However, on occasion diphtheroids may produce endocarditis or urinary tract infection, usually in persons with decreased resistance to infection. Various aerobic and anaerobic gram-positive rods such as Propionibacterium, Lactobacillus, and Listeria, morphologically resemble corynebacteria on Gram stain and are frequently included together in the name diphtheroids without being further identified.

    Actinomyces

    The genus Actinomyces contains several species, of which Actinomyces israelii is the most important. This is a gram-positive filamentous branching bacterium that segments into reproductive bacillary fragments. The organism prefers anaerobic conditions with increased carbon dioxide, but some strains are microaerophilic. Actinomyces israelii produces actinomycosis, a chronic internal abscess-forming infection that characteristically develops sinus tracts to the skin. There typically is a purulent exudate containing small yellow granules, called sulfur granules, which are masses of actinomycetes. Actinomycetes are normal inhabitants of the human oropharynx. Most infections occur in the neck and chest, but infection may occur elsewhere. Several reports indicate a linkage between pelvic infection by A. israelii (which ordinarily is not common) and intrauterine contraceptive devices (IUDs). It has been reported that 5%-10% of asymptomatic IUD users and 40% of IUD users with pelvic infection demonstrate A. israelii on vaginal smear. Culture has not been very successful in isolating the organisms, at least not culture of vaginal specimens. Sulfur granules are rare in vaginal smears. Fluorescent antibody studies on vaginal smears have been more reliable than Papanicolaou cytology smears, although some cytology experts have a high success rate.

    Laboratory diagnosis of nonvaginal actinomycosis consists of Gram stain and acid-fast stain on smears of purulent material containing sulfur granules. The granules should be crushed and smeared. Gram-positive filaments without spores could be either actinomycetes or Nocardia asteroides. Nocardia is acid fast, whereas Actinomyces is not. When material for smear or culture is obtained, special precautions should be taken to avoid contamination by normal oropharyngeal or skin bacteria, which may include nonpathogenic Actinomyces species. A. israelii can be cultured under anaerobic conditions using ordinary anaerobic media. If thioglycollate is used, it should be the enriched type and should be incubated in an increased carbon dioxide atmosphere. Rarely, staphylococci or Pseudomonas bacteria may produce infection with formation of sulfur granules, a condition known as botryomycosis.

    Actinomyces was originally thought to be a fungus, and actinomycosis has many clinical characteristics of a deep fungal infection. Actinomycosis is often included in the differential diagnosis of fungal diseases.

    Nocardia

    The genus Nocardia has similarities to Actinomyces, since the members of both genera are gram-positive filamentous branching bacteria that segment into reproductive bacillary fragments. However, Nocardia is aerobic rather than anaerobic, is weakly acid-fast, and usually does not form sulfur granules. The organisms are saprophytes and are found in soil, grasses, grains, straw, and decaying organic matter. The major pathogenic species (80%-90% of Nocardia infections) is Nocardia asteroides. The human infections produced are primary skin lesions (mycetomas), which are very uncommon in the United States, and visceral infections. Of the visceral infections, the lungs are involved in 60%-80% of cases, and the brain is affected in 20%-40%. In the lung, the most common disease produced is focal pneumonia, which often progresses to abscess formation (frequently multiple). However, various x-ray findings have been reported. Pulmonary nocardiosis frequently resembles TB. Occasionally the infection becomes disseminated. There is no preexisting disease in about 25%-35% of cases (range, 15%-71%). About 25% are associated with malignancy (range, 0%-48%). Some series found association with some type of immunosuppression in 50% or more (chronic infection, TB, steroids, diabetes, AIDS, etc.). Nocardia braziliensis is the most common cause of the chronic focal skin and subcutaneous infection known as mycetoma.

    Culture is the usual method of diagnosis. The organism grows on various media. However, colonies may take 3 days or more to appear so that cultures on ordinary bacteria media may be discarded before being detected. If cultures are placed on mycobacterial culture media, Nocardia may be mistaken for a rapid-growing nontuberculous Mycobacterium. The weak acid-fast stain reaction may reinforce this impression.

    Chlamydiae

    Chlamydiae (formerly called bedsoniae) are tiny coccobacillary organisms classified as bacteria because they have a cell wall, are capable of proteinsynthesis, contain both DNA and RNA, and can reproduce by fission. On the other hand, they have some features of a virus in that they are obligate cell parasites, cannot synthesize adenosine triphosphate (ATP), must depend on host cell metabolic processes for energy, and cannot be grown on artificial laboratory media, only in living cells.

    There are three species of Chlamydia. One is C. psittaci, the etiologic agent of psittacosis in birds and humans. The second is C. pneumoniae, which causes pharyngitis and pneumonia. The third is C. trachomatis. C. trachomatis includes several serotypes that can be placed into three groups. One group causes lymphogranuloma venereum, a venereal disease. A second group causes the eye disease known as trachoma, and the third group produces genital tract and certain other infections, the genital infections being different from lymphogranuloma venereum.

    Chlamydia Psittaci

    This produces psittacosis in humans through contact with infected birds, most often parakeets, or as an occupational disease from infected turkeys in the poultry-processing industry. Only a few cases are reported each year in the United States. There are two types of clinical illness: a respiratory type resembling “atypical” pneumonia and a much less common typhoidlike illness. The respiratory type is characterized by headache, chills, fever, and nonproductive or poorly productive cough. There may be myalgias, arthralgias, GI tract symptoms, bradycardia, and changes in mental status. Splenomegaly is found in many patients. The WBC count is normal or slightly decreased. There are various patterns of lung infiltrates on chest x-ray film, although the most typical are patchy lower lobe infiltrates radiating from the hilar areas. Diagnosis can be made by culture, although this is rarely done since tissue culture is required. Diagnosis is usually made by serologic testing of acute- and convalescent-stage serum.

    Chlamydia pneumoniae

    C. pneumoniae was originally classified as the TWAR strain of C. psittaci but now is a separate species. About 30%-50% (range, 50%-75%) of adults have antibodies derived from previous infection, and C. pneumoniae is said to cause about 10% of pneumoniae and 10%-20% of “primary atypical pneumonia” (pneumonia producing sputum containing WBCs but without bacterial pathogens on standard bacterial culture). This type of pneumonia is similar to that caused by Mycoplasma pneumoniae or Legionella pneumoniae. C. pneumoniae pneumonia is uncommon under age 5 years. Up to 70% of infections are either asymptomatic or afebrile. Some patients react with asthmalike symptoms. Reinfection is said to be common.

    Diagnosis is primarily made through serologic tests. Culture can be done but requires a special cell culture line, takes several days, and is relatively insensitive. The basic original serologic test was complement fixation (CF), which is specific for Chlamydia genus but not for species, has sensitivity only about 50%, and usually requires acute and convalescent specimens for definite diagnosis. This was mostly replaced by a specific microimmunofluorescent (MIF) test using antigen from tissue culture. Although little information is available on % MIF sensitivity in C. pneumoniae disease (due to lack of good gold standards), it appears to be substantially more sensitive than culture and might be in the range of 80%-90% of the sensitivity that is obtained in a similar test method used with C. trachomatis infection. At present, this test is available primarily in large reference laboratories or university centers. Specific nucleic acid probes have also been reported and may have even a little better sensitivity than MIF. These too, at present, would need referral to reference or university laboratories.

    Chlamydia trachomatis

    Lymphogranuloma venereum. Chlamydia trachomatis produces a distinctive venereal disease known as lymphogranuloma venereum, which is transmitted through sexual intercourse. After an incubation period, the inguinal lymph nodes become swollen and tender in males; in females, the lymphatic drainage is usually to the intraabdominal, perirectal, and pelvic lymph nodes. In a considerable number of cases, the perirectal nodes develop abscesses, and the nearby wall of the rectum is involved, eventually leading to scar tissue and constriction of the rectum. In either male or female, in acute stage of lymphatic involvement there may be fever, malaise, headache, and sometimes joint aching, but all of these may be absent. In the majority of cases the main findings are acute inguinal node enlargement in the male and chronic rectal stricture in the female.

    Laboratory findings and diagnosis. Laboratory findings in active disease include mild anemia, moderate leukocytosis, and serum protein hypoalbuminemia and hypergammaglobulinemia. On serum protein electrophoresis the gamma-globulin fraction is often considerably elevated without the homogeneous sharp spikelike configuration of monoclonal protein. Similar polyclonal configurations can be found in granulomatous diseases such as TB or sarcoidosis, in some patients with cirrhosis, and in some patients with rheumatoid-collagen disease.

    Laboratory diagnosis is usually made through a CF serologic test. The CF reaction becomes positive about 1 month after infection and remains elevated for years. There is also a microimmunofluorescence serologic test that is said to be more sensitive than CF. Acute and convalescent serum specimens should be obtained to demonstrate a rising titer. Relatively low positive CF titers (up to 1:64) may occur in persons with genital tract infections due to serotypes or strains of C. trachomatis that do not cause lymphogranuloma venereum. Also, the test cross-reacts with psittacosis, although this is usually not a problem. It is possible to aspirate affected lymph nodes for culture (although tissue culture is required) and for smears with Giemsa stain or immunofluorescent stains, although reports indicate that positive results are obtained in less than one third of the cases. At one time a skin test known as the Frei test was the mainstay of diagnosis, but the antigen is no longer commercially available. Lymph node biopsy shows a characteristic histologic pattern, which, however, is also seen in tularemia and cat scratch fever. Lymphogranuloma venereum is associated with a relatively high incidence of syphilis serology biologic false positive reactions. This may be difficult to interpret, because syphilis must be suspected in any patient with venereal disease, and early syphilis can produce inguinal lymph node enlargement similar to that caused by lymphogranuloma venereum.

    Chlamydial urethritis and endocervicitis. Chlamydia trachomatis can also produce urethral and genital infections that differ clinically from lymphogranuloma venereum and are associated with different organism serotypes. C. trachomatis is reported to cause about 50% (range, 30%-60%) of nongonococcal urethritis in men. Several studies of clinically normal sexually active men not at high risk for venereal disease report asymptomatic chlamydial infection rates of about 10% (range, 5%-26%). About one third of male urethral chlamydial infection is asymptomatic. In some geographic areas or patient populations the incidence of nongonococcal urethritis is two to three times that of gonorrhea. Male patients with gonococcal infection also have chlamydial infection in 20%-30% of cases; and about 70% of cases of postgonococcal urethritis have been attributed to Chlamydia. The urethral discharge in nongonococcal urethritis is usually less purulent than the discharge produced by gonorrhea. C. trachomatis has been found in 40%-50% (range, 6%-69%) of patients with Reiter’s syndrome. Finally, male sexual consorts of women who have chlamydial cervicitis or chlamydial PID are themselves infected by Chlamydia in 25%-50% of cases.

    Chlamydiae can infect the female urethra and also the endocervix. Exact incidence in the normal population is unknown, but it has been cultured from the endocervix in about 10% (range, 2%-30%) of asymptomatic women not thought to have venereal disease, 8%-26% of girls attending adolescent clinics, 6%-23% of women attending contraception clinics, in 27%-37% of pregnant female adolescents, and in 10%-48% of women seen in venereal disease clinics. About 60%-80% of infected women are asymptomatic. About 75% of infected women have endocervical infection, about 50% have urethral infection, and about 25% have rectal infection. About 60%-80% of women with cervical infection by Chlamydia are asymptomatic. Patients with PID have a 20%-30% rate of chlamydial recovery from the endocervix and from the fallopian tubes or pelvic abscesses. About 30%-50% of women with gonococcal infections also have chlamydial infection. Chlamydia is also reported to be a major cause for culture-negative female dysuria with frequent urination (acute urethral syndrome), described earlier in this chapter. Finally, female sexual consorts of men who have proven chlamydial urethritis are themselves infected by Chlamydia in up to 70% of cases. If the male consort has gonorrhea, female infection rate for Chlamydia is about 35%; if the male has nongonococcal urethritis, the female will have chlamydial infection in 30%-40% of cases. Chlamydia is now the most common sexually transmitted disease in the United States, causing more infections yearly than all other etiologies combined.

    Diagnosis. Diagnosis of C. trachomatis urethritis or cervicitis has been attempted in several ways. Culture (McCoy cell tissue culture) is still considered the gold standard. In males, a thin nasopharyngeal-type calcium alginate swab is inserted 3-4 cm into the urethra. The swab should be rotated for at least 5 seconds and at least one revolution. In females, Chlamydia infects only the columnar and squamocolumnar cervical cells, so that endocervical specimens produce maximum isolation rates, whereas vaginal specimens are not recommended. Therefore cervical culture specimens in the female should be obtained 1-2 cm above the squamocolumnar junction, into the endocervix. The swab should be rotated against the endocervix for 10-30 seconds. Gently cleaning the endocervical canal with a swab before taking the specimen is reported to decrease cell culture contamination and increase positive results. Sensitivity of culture using one swab has varied from 33%-86%. Obtaining two swabs instead of one is reported to increase positive yield from 5%-45%. In one study, an additional urethral culture increased sensitivity by 23%. Swabs with a wooden handle should not be used because the wood contains chemicals that inhibit chlamydiae. Immediately after the specimen is obtained, special transport media should be innoculated with the swabs. The innoculated transport media should be kept at 4°C if culture can be done within 24 hours. If culture cannot be done until after 24 hours, its specimen should be stored at –70°C. Storage in a “frost-free” freezer or at –20°C temperature decreases culture yield. A specimen collection brush (Cytobrush) was claimed to considerably increase the number of columnar cells and thereby increase satisfactory cultures or direct smears in one study but had equal sensitivity to swabs in two other studies. Sensitivity of culture is estimated to be about 80%-90% (range, 63%-97%).

    Several manufacturers have marketed kits for direct fluorescent antibody (FA) detection of Chlamydia organisms within infected cells, using the same specimens obtained for culture applied directly to a slide or microtiter wells. This permits same-day results without the delay and many of the technical problems of tissue culture. Only two of these (FA) systems have had reasonably adequate evaluation. Their sensitivity has averaged about 80%-85% (for one of these, the range has been 60%-96%) compared to culture (since culture sensitivity is not 100%, true sensitivity of the FA methods is less than the stated figures). Specificity has averaged 97%-98%.

    Some investigators recommend that the direct tests not be used on low-prevalence (<10% incidence) populations, since in that patient group, although the percentage of false positive test results is low, in terms of the number of patients the false positive results become too high. In addition, the percentage of false positive and negative results using FA methods would probably be higher in ordinary laboratories than in research laboratories. Enzyme immunoassay (EIA or ELISA) kits have been replacing FA. Average sensitivity of these EIA kits is about 75%-85% (range, 44%-100%), about the same as FA. One EIA kit evaluation reported detection of over 90% of patients using urine specimens (in males) but only 66% using blood specimens from the same patients. A commercially available DNA probe also has about the same sensitivity (75%-85%; range, 60%-97%). Several nucleic acid probes with PCR amplification have been evaluated; all were more sensitive than culture (about 1.5 times more than culture; range, 1.04-4.5 times). One report indicates that one commercial probe with PCR amplification detected 95% of male urethral chlamydial infections using a urine specimen.

    Other chlamydial infections

    About 5%-10% (range, 2%-25%) of pregnant women have Chlamydia infestation of the cervix. Fifty percent to 70% of infants born to these infected mothers acquire Chlamydia infection or colonization during birth. Of these, about 30%-50% (range, 18%-75%) develop chlamydial conjunctivitis, with onset about 1-3 weeks after birth; and about 10%-15% (range, 3%-30%) develop chlamydial pneumonia, with onset about 4-12 weeks after birth. In addition, it has been reported that asymptomatic colonization of the vagina, rectum, and pharynx, sometimes lasting over 2 years, may occur in 15% of infected infants. According to some reports, chlamydial infection causes 25%-80% of infant conjunctivitis and about 30% of infant pneumonia. Diagnosis can be made by culture or FA methods from swab material obtained from the posterior nasopharynx, similar to specimens for pertussis. Special transport media are necessary for culture specimens. In possible chlamydial conjunctivitis, culture specimens can be obtained or smears can be made from conjunctival scrapings. Purulent material should be wiped off before scrapings or cultures are obtained since the organisms are found in conjunctival epithelial cells and not in the inflammatory cells of the exudate. Giemsa-stained smears have been used for many years but are difficult to interpret and detect only about 25%-40%.

    Trachoma. Finally, C. trachomatis produces trachoma, a serious eye disease that is endemic to North Africa, the Middle East, and Southeast Asia. The conjunctiva is infected and becomes scarred. The cornea is eventually invaded by granulation tissue and is destroyed. Diagnosis usually is made from Giemsa-stained smears of conjunctival scrapings. The infected cells contain cytoplasmic inclusion bodies. However, results of only about one half of the cases are positive. Immunofluorescent techniques applied to the smears produce positive results in about 85% of cases. McCoy cell culture detects about 95% of cases.

    Mycoplasma

    Mycoplasmas are similar to bacteria in most respects and can be grown on artificial media. However, they lack a cell wall and are smaller than bacteria. There are several genera in the Mycoplasmataceae family. One of these is Ureaplasma, which contains a species, Ureaplasma urealyticum (formerly known as T. mycoplasma) that has been cultured from the urethra of both sexes and cervix or vagina of 40%-80% of clinically normal females. The incidence of positive cultures is increased in persons seen in venereal disease clinics. It is thought that U. urealyticum is an important cause of nongonococcal urethritis in men.

    There is accumulating evidence that U. urealyticum can produce placental and fetal infection in obstetrical patients already colonized with the organism. Transmission to the fetus, placenta, or fetal membranes (vertical transmission) is reported to occur in 45%-66% of full-term infants. In most, this results in temporary colonization, with 10% or less of older children still culture-positive. However, in a small number there may be chorioamnionitis, spontaneous abortion, premature birth; less commonly there may be other conditions such as neonatal pneumonia and bronchopulmonary dysplasia. Since maternal colonization is so frequent, this colonization by itself does not predict pregnancy or fetal complications. Diagnosis of infection or suspected infection is usually made by culture. A noncotton swab (or tissue biopsy) should be innoculated directly into special ureoplasma transport medium and the swab then removed. Blood (as much as possible) without anticoagulants can be innoculated into the transport medium in a 1:19 (blood to medium) ratio. Specimen should be refrigerated or kept cold until they enter the laboratory. If the specimen must be sent to a reference laboratory, it should be frozen (at –70°C if possible) and sent frozen.

    Another genus has the name of Mycoplasma and contains two species associated with human disease, Mycoplasma hominis and Mycoplasma pneumoniae. Mycoplasma hominis, like U. urealyticum, is a normal inhabitant of the female urethra and female genital organs (20%-40% of persons) and to a lesser degree in the male urethra. In addition, it is occasionally found in the oropharynx of both sexes. The role of M. hominis in nongonococcal urethritis is not clear, but it currently is not thought to produce many cases, if any. There is more evidence for occasional involvement in female PID and occasional infections in immunocompromised patients.

    Mycoplasma pneumoniae. Mycoplasma pneumoniae (formerly called Eaton agent, pleuro-pneumonia-like organism) is the most frequent etiologic agent of a lower respiratory tract infection formerly called primary atypical pneumonia (pneumonia with sputum containing neutrophils but no bacterial pathogen cultured). However, Legionella pneumophilia, Chlamydia pneumoniae, and viruses can also produce this syndrome. In one study, Mycoplasma pneumophilia was cultured in 13% of patients with nonstreptococcal acute pharyngitis. Some investigators report that mycoplasma caused 20%-25% of all cases of community-acquired pneumonia in their area. Clinical mycoplasma pneumonia is commonly seen only between the ages of 5-40 years. Patients can be reinfected after 2-10 years. Most cases appear as an upper respiratory infection of varying severity. If pneumonia develops, it is usually preceded by headache, fever, and malaise. Sore throat or chills occur in 30%-50% of patients, and GI symptoms or earache in a lesser number. After 2-4 days there is onset of a nonproductive or poor productive cough. Chest x-ray typically shows lower lobe mottled infiltrates that are most pronounced in the hilar region and that are most often unilateral. However, in some cases other abnormalities develop. Despite the symptoms and chest x-ray findings there is characteristically little abnormality on physical examination.

    Laboratory findings and diagnosis. WBC counts are usually normal, although about 25% of affected persons have mild leukocytosis (between 10,000 and 15,000/mm3; 10-15 x 109/L). An unusually severe case may produce more abnormal results. Several modalities are available for diagnosis. Culture can be done using sputum or nasopharyngeal swabs. Mycoplasma pneumoniae is very sensitive to drying, so the swab must be placed immediately in a special transport medium. Special culture media are required, and most laboratories must send the specimen to a reference laboratory. Such specimens should be frozen and transported with dry ice. Growth takes about 2 weeks (range, 1-3 weeks). Compared to CF in various studies, isolation rates from sputum or throat swabs averages about 50%-70% (range, 0%-87%), so that culture does not seem clinically very helpful.

    Cold agglutinins are elevated in 50%-60% of patients. Cold agglutinins are antibodies that are able to agglutinate type O human blood cells at refrigerator temperatures but not at room temperature. They are found in other diseases and thus are nonspecific; but in adults with an acute respiratory syndrome, their presence in significant titer is usually associated with mycoplasmal pneumonia. Cold agglutinin titers elevated more than 1:32 are abnormal and can be found during the second week of illness, reaching a peak in the third or fourth week. A rising titer is more significant than a single determination.

    Serologic tests are the most common method of diagnosis. Mycoplasmal CF test results are abnormal in 60%-70% of cases (range, 45%-80%). Antibody becomes detectable during the second week of illness and peaks in the fourth week. The CF reaction may become nondetectable after 5-8 years. Immunofluorescent methods for antibody detection are replacing the cumbersome and time-consuming CF technique. Immunofluorescent methods that can detect either IgM or IgG antimycoplasmal antibodies are available (sensitivity 80%-85% compared to CF). The presence of IgM antibodies, especially if present in high titer, usually means acute or recent infection. Immunoglobulin G antibodies rise later than IgM antibodies and persist much longer. It is useful to obtain two serum specimens with a 1-week interval between the first and the second with either IgM or IgG methods. Low levels of mycoplasmal IgG antibodies are very frequent in the general population. Latex agglutination kits are available from several companies; most detect total antibody (IgM plus IgG). Sensitivity varies depending on whether culture or CF tests are used as the gold standard, with reported sensitivity compared to CF about 90% (range, 53%-98%). Finally, one company has marketed a DNA probe method for Mycoplasma that can be applied directly to throat swab specimens, can be performed the same day, and is reported to give 90%-100% correlation with culture. Drawbacks are relatively high cost if only one patient specimen is processed at a time plus a significant but unknown percent of false negative results if detection rates are no better than culture. Other reports have evaluated DNA probes, mostly using home made reagents, which were compared to several different gold standards, thus adding to the variability introduced by different stages of infection and mixes of patients to the variability guaranteed by different antibodies and techniques.

    Legionella

    Members of this genus are tiny gramegative rods that require special media for culture. They seem to be rather widely distributed and in some cases are linked to water habitats. Most are associated with respiratory tract infection. The predominant species is Legionella pneumophilia; of this species there are two basic types of clinical illness. One is an influenza-like form (sometimes called Pontiac fever), which is nonfatal; and the other is the “primary atypical pneumonia” (classic “Legionnaire’s disease”) form that has been fatal in 10%-20% of cases. Other species have been identified (e.g, Legionella micdadei, the etiology of Pittsburgh pneumonia; see Table 37-9). There is an increased incidence of L. pneumophilia infection in patients who are immunocompromised or have conditions associated with decreased resistance to infection. Spread of infection has most frequently been associated with water in cooling systems.

    In the pneumonic Legionnaire’s disease form of illness, there typically is a prodromal stage of 1-10 days with influenza-like symptoms (headache, fatigue, myalgias, and sometimes chills). After this, pneumonia develops with high fever (<103°F; <39.4°C) in approximately 70% or more of cases, recurrent chills in about 70%, nonproductive or poorly productive cough in 50%-80%, pleuritic chest pain in 15%-40%, pleural effusion in 15%-40%, relative bradycardia in approximately 50%, toxic encephalopathy (confusion, disorientation) in 35% or more, and diarrhea in 20%-50%. Laboratory abnormalities include leukocytosis (10,000-30,000/mm3) in 60%-80% of affected persons and mild liver function test elevations in approximately 30%-40%. Proteinuria occurs in about 40% of cases and microscopic hematuria in about 10%. In some series 50% or more of patients had hyponatremia and hypophosphatemia. Radiologically there is pneumonia, which begins unilaterally in 50%-70% of cases, and consists of patchy bronchopneumonia or small densities. This progresses to lobar pneumonia in up to two thirds of patients in some reports, which frequently becomes multilobar and which may become bilateral. Some patients have lobar pneumonia when they are first seen.

    Diagnosis is made by culture on special media (not often available in routine laboratories). Although Legionella can be cultured from sputum, there frequently is little sputum produced, and there may be too much contamination by oropharyngeal bacteria. However, one report indicates that rejecting a sputum specimen for Legionella culture on the basis of usual criteria for contamination based on number of squamous epithelial cells or for noninfectivity based on its number of WBCs would result in missing 47%-84% of Legionella culture-positive cases (depending on rejection criteria used). Yield is generally much better from transtracheal aspiration, bronchial washings, pleural fluid, and lung biopsies. Sensitivity of culture presently is about 70% (range, 15%-80%), and the procedure usually takes 3-5 days. Specimens should be kept moist using a small amount of sterile water; saline inhibits growth of Legionella. The specimen should be refrigerated if it cannot be delivered with 30 minutes. Direct fluorescent antibody (DFA) stain on smears prepared from respiratory tract material can be performed in 1-2 hours. However, sensitivity on these specimens is only about 50%-60% (range, 20%-75%). Urine antigen in Legionella disease becomes detectable in 80% of patients at day 1-3 of clinical illness and persists for varying time periods, occasionally as long as a year. Two commercial companies market a latex agglutination kit for urinary antigen (the two kits are apparently identical). In two evaluations, sensitivity was reported to be 54%-92% and specificity was 46%-74%. ELISA methods have been reported to be 70% or more sensitive in detecting urine antigen. Serologic testing (generally by indirect fluorescent antibody methods) is still used, especially for epidemiologic investigation. A fourfold rise in titer to at least 1:128 is diagnostic, and a single titer of at least 1:256 is considered presumptive evidence when combined with appropriate symptoms. However, only about 50% of patients seroconvert by the second week of clinical illness, and it may take 4-6 weeks to obtain maximal rates of seroconversion. Ten percent to 20% of patients do not develop detectable antibodies. Nucleic acid (DNA) probe techniques are beginning to be introduced. At least two of these are said to detect about 67%-74% of cases when applied to respiratory material, which is an increase in sensitivity over DFA. However, DNA probe is expensive and is very expensive when only single specimens are processed.

  • Nontuberculous Mycobacteria

    There are other mycobacteria besides Mycobacterium tuberculosis, some of which are frequently pathogenic for humans and some of which rarely cause human infection. The nontuberculous mycobacteria were originally called “atypical mycobacteria.” The first useful classification was that of Runyon, who subdivided the nontuberculous mycobacteria into four groups, depending on growth speed and colony characteristics (Table 14-2). These groups have some clinical value as rough guides to the type of organism present while awaiting more definitive identification and speciation. It is desirable to place the organisms in the correct species, since some members (species) of any of the Runyon groups may not be pathogenic very often or may differ in degree of pathogenicity. Mycobacterium intracellulare or Mycobacterium avium-intracellulare complex (formerly known as the Battey Mycobacterium), a member of Runyon group III, and Mycobacterium kansasii, from Runyon group I, together cause the majority of significant nontuberculous mycobacterial infections in about equal proportions. These produce a disease similar to pulmonary tuberculosis (although often milder or more indolent) that is much more frequent in adults. M. avium-intracellulare infections are very frequent in persons with acquired immunodeficiency syndrome (AIDS) or AIDS-related conditions. The mycobacterial infection frequently becomes bacteremic or disseminated due to compromise of the immune system by HIV-1 causing AIDS. Runyon group II organisms are more frequent in children and clinically tend to cause cervical lymphadenopathy. Diagnosis of the nontuberculous mycobacteria is essentially the same as for M. tuberculosis. Skin tests (old tuberculin or PPD) for M. tuberculosis will also cross-react with the nontuberculous mycobacteria. In general, the nontuberculous mycobacteria tend to produce less reaction to standard tuberculin skin tests than M. tuberculosis. In fact, several studies claim that the majority of positive intermediate-strength tuberculin skin test results that produce a reaction of less than 10 mm diameter are due to nontuberculous mycobacterial infection rather than TB. Skin test antigens are available for each of the nontuberculous mycobacterial groups, although some reports challenge the specificity of these preparations. The main clinical importance of these nontuberculous organisms is resistance that many have toward one or more of the standard antituberculous chemotherapeutic agents.

    Classification of the atypical mycobacteria

    Table 14-2 Classification of the atypical mycobacteria

    Several reports have linked one of the non-MTB organisms, M. paratuberculosis, to Crohn’s disease (regional ileitis).

  • Tuberculosis and Mycobacterial Disease

    Tuberculosis is caused by Mycobacterium tuberculosis (MTB), a rod-shaped bacterium that requires special media for culture and that has the peculiarity of “acid-fastness” (resistance to decolorization by strong acidic decolorizing chemicals such as acid alcohol after being stained by certain stains such as carbol fuchsin). Tuberculosis is still very important and common despite advances in drug therapy. It has been reported that about 25% of persons exposed to MTB will become infected; and of those infected, about 10% will develop clinical disease (range, 5%-40%). The disease usually begins in the chest due to inhalation of airborne infectious material. This material is carried to some localized area of the lung alveoli and provokes a host response of granulomatous inflammation around the material (the “Ghon complex”). It is thought that in many cases there is also a silent hematogenous spread of the organisms. In most cases the host is able to contain and eventually destroy the organisms in the chest and those reaching other locations. Those in the lungs seem better able to survive than those deposited elsewhere. In some cases the organisms remain dormant, and the infection can be reactivated at a later date; in some cases the initial infection spreads; and in some cases reinfection takes place. If infection in the lungs progresses to clinical disease, the most important symptoms are cough, fever, and hemoptysis. (The most important diseases to rule out are lung carcinoma and bronchiectasis.) The kidney is involved in a small percentage of advanced cases, with the main symptom being hematuria (Chapter 12). A small number of patients develop widespread extrapulmonary disease, known as miliary tuberculosis. The laboratory findings in tuberculosis depend to some extent on the stage and severity of the disease.

    Chest x-ray films

    Chest x-ray films often provide the first suggestion of tuberculosis and are a valuable parameter of severity, activity, and response to therapy. Depending on the situation, there are a variety of possible roentgenographic findings. These may include one or more of the following:

    1. Enlargement of hilar lymph nodes.
    2. Localized pulmonary infiltrates. These occur characteristically in an upper apical location or, less commonly, in the superior segment of the lower lobes. Cavitation of lesions may occur.
    3. Miliary spread (small punctate lesions widely distributed). This pattern is not common and may be missed on routine chest x-ray films.
    4. Unilateral pleural effusion. The most common causes are tuberculosis, carcinoma, and congestive heart failure. Tuberculosis has been reported to cause 60%-80% of so-called idiopathic pleural effusions, although this percentage varies greatly depending on the patient’s geographic location and other factors.

    Sputum smear

    Sputum smears provide a rapid presumptive diagnosis in pulmonary tuberculosis. The smear is usually stained by one of the acid-fast (acid-fast bacillus, or AFB) procedures (usually the Ziehleelsen or Kinyoun methods). Fluorescent Auramine-o staining methods are available, faster, and somewhat more sensitive. Smears require about 5 Ч 103 organisms/ml of specimen for microscopic detection. The more advanced the infection, the more likely it is to yield a positive smear. Therefore, the rate of positive findings is low in early, minimal, or healing tuberculosis. Also, the smear may be normal in a substantial minority of advanced cases. Culture is more reliable for detection of tuberculosis and also is necessary for confirmation of the diagnosis, for differentiation of MTB from the “atypical” mycobacteria, and for sensitivity studies of antituberculous drugs. According to the literature, false negative smears (smear negative but culture positive) have been reported in an average of 50% of cases (literature range, 16%-70%). Some of these false negative results may be due to laboratory technique problems and differences in smear staining methods. A high centrifugation speed when concentrating the specimen is said to increase the yield of positive smears. False positive smears (positive smear but negative culture) have been reported, averaging about 1%-5% of positive smears (literature range, 0.5%-55%). Some of these were apparently due to contamination of water used in the smear-staining procedure by saprophytic mycobacteria. Control slides are necessary to prevent this. Some authorities believe that only 1-2 acid-fast organisms/300 oil immersion fields should be considered negative (although indicative of need for further specimens). Smears may sometimes be positive for up to 2 months when cultures are negative if the patient is on antituberculous drug therapy (this would not be considered a genuine false positive, since the drugs inhibit mycobacterial growth or the organisms may be nonviable). After 2 months, persistence of positive smears raises the question of treatment failure. Temporary persistence of positive smears with negative cultures is more likely to occur in severe cavitary disease (in one series, this occurred in 20% of cases). Sputum specimens should be collected (for culture and smear of the concentrated specimen) once daily for at least 3 days. If the smear is definitively positive, further smears are not necessary. Also, a definitively positive smear means high probability that culture of the specimens already collected will obtain positive results, and it is not necessary to collect more than three specimens or to proceed to more complicated diagnostic procedures. If smears are negative, one must consider the possibility that the culture may also be negative, and conventional cultures on standard solid media average 20 days to produce growth from MTB smear-positive specimens and about 27 days from MTB smear-negative specimens (overall range, 2–8 weeks).

    Culture

    Sputum culture is preferred for pulmonary tuberculosis (gastric aspiration may be done if adequate sputum specimens cannot be obtained); urine culture is preferred for renal involvement; and bone marrow culture is preferred in miliary tuberculosis. Reports indicate that an early morning specimen, either of sputum or urine, produces almost as many positive results as a 24-hour specimen and has much less problem with contamination. Special mycobacteria culture media are needed. The necessity for adequate sputum culture specimens, regardless of the concentrated smear findings, should be reemphasized. Several reports indicate that aerosol techniques produce a significantly greater yield of positive cultures than ordinary sputum collection. The aerosol mixture irritates the bronchial tree and stimulates sputum production. At any rate, it is necessary to get a “deep cough” specimen; saliva alone, although not completely useless, is much less likely to reveal infection and is much more likely to be contaminated. If sputum cultures are negative or if the patient is unable to produce an adequate sputum sample, gastric aspiration may be used. Gastric contents are suitable only for culture; nontuberculous acid-fast organisms may be found normally in the stomach and cannot be distinguished from M. tuberculosis on AFB smear. If renal tuberculosis is suspected, urine culture should be done (Chapter 12). However, renal tuberculosis is uncommon; and even with urine specimens obtained on 3 consecutive days, only about 30% of cases are positive.

    Cultures should be grown in high carbon dioxide atmosphere, since this is reported to increase the number of positive cultures by at least 10%. Inoculation on several varieties of media increases the number of positive results by 5%-10%. The 4% sodium hydroxide traditionally used to digest and decontaminate sputum before concentration also kills some mycobacteria. Use of weaker digestion agents increases culture yield, but troublesome overgrowth by other bacteria may also increase.

    Culture should be done on all tissue specimens when tuberculosis is suspected. Acid-fast stains on tissue slides reveal tuberculosis organisms in only 30%-40% of cases that are positive by culture. Several newer methods such as BACTEC (which uses liquid media and a machine that detects metabolic products of bacterial growth) have been able to decrease detection time for MTB smear-positive specimens to 8 days and time for MTB smearegative specimens to 14 days (overall range, 1-3 weeks). The system is about 93% sensitive compared to conventional multimedia culture. Once culture growth occurs, the organism must be identified. Conventional methods require biochemical and growth tests to be performed that may take 3-6 weeks to complete. The BACTEC system has a nucleic acid phosphate method that can identify MTB (only) in 3-5 days. Commercial DNA probes are available that can identify MTB and certain non-MTB mycobacteria in 1 day. Gas-liquid chromatography and high-performance liquid chromatography have also been used. Antibiotic sensitivity studies are recommended when a mycobacterial organism is isolated, since multiresistant MTB is increasing in various areas and non-MTB mycobacteria have often been multiresistant. Conventional culture methods take 21 days; BACTEC takes about 5 days.

    Data on sputum culture sensitivity using conventional AFB media is difficult to find since culture is usually considered the gold standard of AFB detection. Sputum culture appear to average about 75% sensitivity (range, 69%-82%). Sensitivity using BACTEC averages about 85% (range, 72%-95%).

    Nucleic acid probe

    Nucleic acid (DNA) probe methods are now becoming available that permit direct nonsmear detection of mycobacteria in clinical specimens. The first such test available (Gen-Probe, Inc.) is reported in two studies to be 83%-85% sensitive compared with culture using sputum specimens. However, it has been reported that antituberculous drugs can interfere with the probe; this study found sensitivity in nontreated patients to be over 90% (when comparing probe to culture, culture only detects about 75%-80% of cases). Ten percent of specimens were positive by probe but negative by culture, which may represent additional true positives that could not be confirmed. A DNA probe is also available specifically for M. tuberculosis. Same-day results can be obtained. Disadvantages of this first-generation method are need for considerable technologist time and certain special equipment. Compared with culture, the general Mycobacterium screen probe will not differentiate M. tuberculosis from other mycobacteria, whereas the specific M. tuberculosis probe will not detect the other mycobacteria. Neither probe would provide therapeutic drug sensitivity information. The major drawback regarding its use as replacement for the acid-fast smear is the relatively high cost of the probe method, very high if only one specimen at a time is processed. DNA probes with PCR amplification have been reported (e.g., Roche Diagnostics) that are said to have a sensitivity of 3-30 organisms/ml (compared to at least 5 Ч 103 organisms/ml required for a positive acid-fast smear). Nevertheless, one study involving 7 prestigious worldwide reference laboratories who were sent sputum or saliva specimens to which various quantities of BCG (M. Bovis) mycobacteria were added, showed false positive PCR rates of 3%-77%. In specimens containing 102 organisms, sensitivity ranged from 0%-55%; in specimens containing 103 organisms, 2%-90%; and in specimens containing 101 organisms, 20%-98%.

    Skin test (Mantoux test)

    This test is performed with an intradermal injection of purified protein derivative (PPD) or old tuberculin (Table 14-1). A positive result is represented by an area of induration having a specified diameter by 48 hours. The diameter used to be 10 mm but was redefined in 1990 to require different diameters depending on the person’s risk group (see box). In addition, a distinction was made between “reaction” (diameter or width of induration without record of previous test result) and “conversion” (increase in reaction width within 2 years from last previous reaction width). For all persons younger than 35 years of age whose previous reaction was negative, an increase in PPD induration of 10 mm or more in diameter within a period of 2 years would be considered a conversion and presumptive suspicion for occult tuberculosis (TB), whereas the change would have to be at least 15 mm for persons 35 years of age or more (that is, for nonrisk persons above or below age 35 who have had a PPD within 2 years, conversion criteria would replace reaction size criteria).

    Table 14-1 Comparison of tuberculosis skin tests*
    Comparison of tuberculosis skin tests


    A positive skin test is a manifestation of hypersensitivity to the tubercle bacillus. This reaction usually develops about 6 weeks after infection, although it may take several months. A positive reaction means previous contact and infection with TB; the positive reaction does not itself indicate whether the disease is currently active or inactive. However, in children under 3 years of age it usually means active TB infection. Apparently, once positive, the reaction persists for many years or for life, although there is evidence that a significant number of persons revert to negative reactions if the infection is completely cured early enough. In a few cases of infection the test never becomes positive. The Mantoux test may revert to negative or fail to become positive in the following circumstances:

    1. In about 20% of seriously ill patients, due to malnutrition (severe protein deficiency).
    2. In newborns and occasionally in old age.
    3. In some persons with viral infections, or within 1 month after receiving live virus vaccination.
    4. In 50% or more of patients with miliary tuberculosis.
    5. In a high percentage of patients with overwhelming pulmonary tuberculosis.
    6. In a considerable number of patients who are on steroid therapy or immunosuppressive therapy.
    7. In many persons who also have sarcoidosis or Hodgkin’s disease.
    8. In some persons with chronic lymphocytic leukemia or malignant lymphoma.
    9. In some patients with chronic renal failure or severe illness of various types.
    10. In some persons with old infection (“waning” of reactivity).
    11. When there is artifact due to improper skin test technique (e.g, subcutaneous rather than intradermal injection).

    In cachectic patients and those with protein malnutrition, treatment with an adequate protein diet can restore Mantoux test reactivity to most patients after about 2 weeks. In patients after age 50 with M. tuberculosis infection, especially those with old previous infection, the PPD skin test sometimes may slowly decrease in reactivity and eventually become negative. (How often this occurs is controversial; the best estimate seems to be 8%-10%, but studies range from 0.1%-21%, possibly influenced by the elapsed time period since infection and time intervals between testing.) If another skin test is performed, the new skin test itself stimulates body reaction and may restore reactivity (“booster phenomenon”). This phenomenon could simulate a new infection if previous infection were not known, since the next time a skin test is performed the physician would see only conversion of a negative to a positive reaction. Restoration of skin reactivity can take place in only 1 week, so that retesting 1 week after the first negative reaction can usually show whether or not there is potential for the booster reaction. (The 1-week interval would in most cases not be long enough for true conversion in persons with their first infection.) Repeated skin tests will not cause a nonexposed person to develop a positive reaction. Some investigators recommend skin testing with antigens used to demonstrate the presence of skin test anergy (e.g., Candida or Trichophyton antigen) if the Mantoux test is repeatedly negative in a person with substantial suspicion of mycobacterial infection.

    The standard procedure for skin testing is to begin with an intermediate strength PPD (or the equivalent). If the person has serious infection, some clinics recommend starting with a first-strength dose to avoid necrosis at the injection site. A significant minority of patients with tuberculosis (9%-17%) fail to react to intermediate strength PPD; a second-strength dose is then indicated.

    Miliary tuberculosis

    Miliary TB is clinically active TB that is widely disseminated in the body by hematogenous spread. Clinical symptoms are often nonspecific, such as fever, weakness, and malaise. There frequently is an associated condition, such as alcoholism, intravenous (IV) drug abuse, or malignancy, that decreases immunologic defenses. About 20% have a negative tuberculin skin test reaction. About 35% do not show a miliary pattern on chest x-ray film. If routine clinical and culture methods fail, biopsy of bone marrow or liver may be useful. Liver biopsy has a fairly good positive yield (up to 80%), considering that a needle biopsy specimen is such a tiny random sample of a huge organ. However, it is usually difficult to demonstrate acid-fast organisms on liver biopsy even when tubercles are found, and without organisms the diagnosis is not absolutely certain. Bone marrow aspiration is probably the best procedure in such cases. Bone marrow yields much better results for mycobacterial culture than for demonstration of tubercles. Routine marrow (Wright-stained) smears are worthless for histologic diagnosis in TB. Aspirated material may be allowed to clot in the syringe, then formalin-fixed and processed as a regular biopsy specimen for histologic study. Before clotting, some of the aspirate is inoculated into a suitable TB culture medium. It should be emphasized that bone marrow aspiration or liver biopsy is not indicated in pulmonary tuberculosis (since this disease is relatively localized), only in miliary TB.

    PPD Reaction Size Considered “Positive” (Intracutaneous 5 TU* Mantoux Test at 48 Hours)
    5 MM OR MORE
    Human immunodeficiency virus
    (HIV) infection or risk factors for HIV
    Close recent contact with active TB case
    Persons with chest x-ray consistent with healed TB
    10 MM OR MORE
    Foreign-born persons from countries with high TB prevalence in Asia, Africa, and Latin America
    Intravenous (IV) drug users
    Medically underserved low-income population groups (including Native Americans, Hispanics, and African Americans)
    Residents of long-term care facilities (nursing homes, mental institutions)
    Medical conditions that increase risk for TB (silicosis, gastrectomy, undernourished, diabetes mellitus, high-dose corticosteroids or immunosuppression RX, leukemia or lymphoma, other malignancies
    Employees of long-term care facilities, schools, child-care facilities, health care facilities
    15 MM OR MORE
    All others not listed above
    * TU, tuberculin units.

    Renal tuberculosis

    Renal TB is almost always bilateral and presumably results from nonhealing infection produced during the transient bacteremia of the lung primary stage. There is usually a latent period of many years before clinical infection becomes evident. The estimated incidence of eventual active renal infection is about 2%-5%, but this probably represents incidence in high-risk groups. About 25% (range, 20%-75%) of patients are said to have a normal chest x-ray film. About 14% (range, 12%-15%) of patients are reported to have a negative PPD skin test result. Even the intravenous pyelogram results (IVP) are normal in about 25% (range, 14%-39%) of cases. Most patients do not have systemic symptoms, such as fever. The erythrocyte sedimentation rate was elevated in 23% of patients in one report. Only 20%-56% have urinary tract symptoms. Gross hematuria is the classic finding in renal TB but is present in only about 20% of patients. Pyuria (with negative urine culture) and microscopic hematuria are more frequent, occurring in about 65%-85% of cases. Some patients have a positive urine culture with some ordinary pathogen in addition to renal TB. Urine culture for TB was mentioned previously; 30%-80% of patients have positive cultures when three 24-hour or early morning specimens are collected (true culture sensitivity is probably less than 80%, since the diagnosis could easily be missed with negative cultures).