Polish Up on Past Pestilence and Present Pathogens

Written By: Sam Savage

Published Date: July 4, 2008 Last Edited: July 4, 2008

By Schofield, Cynthia B

Smallpox, diphtheria, typhoid fever, polio, cholera, and tuberculosis (TB) are among the diseases that struck fear into citizens of 18th- and 19th-century America. Discovery of antibiotics and antiseptic techniques in 20th-century America made the threat of death from an infectious disease seem like a horror of the past. In 1882, Robert Koch isolated and identified the Mycobacterium tuberculosis (MTB) bacillus, followed by Vibrio cholerae a year later. Louis Pasteur, renowned for his “germ theory” of disease, developed sterilization techniques that revolutionized the practice of medicine. Another early researcher, Edward Jenner, introduced the world to vaccination when he discovered that the cowpox vaccine conferred immunity to the dreaded smallpox disease; smallpox was thought to have been eliminated in 1977.

Beginning with the discovery of streptomycin in the 1940s, the rate of tuberculosis declined as new and more effective antibiotics were developed. Unfortunately, the 1980s brought co-infection with TB to many patients already suffering from HIV/AIDS. Resistant strains of TB also appeared; the multidrug resistant (MDR-TB) strain was followed by extensively-resistant TB (XDR-TB).1 By the 1990s, an escalating incidence of antibiotic resistance in many strains of bacteria was occurring. Nosocomial infection was out of control when resistant organisms began spreading in the hospital environment: methicillin-resistant Staphylococcus aureus (MRSA), vancomycin resistant Entewcoccus spp. (VRE), and Clostridium difficile- associated disease (CDAD).

Despite years of intensive measures by the World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC) to prevent and control the recent transmission of infection, emerging and re-emerging pathogens are creating serious infectious threats in today’s world. The following illustrates their epidemic/pandemic nature and our present limitations for controlling them.

CASE I: Corymbacterium diphtherias – After a recent trip to Haiti where he had helped build a church, a 63-year-old man presented to the emergency department (ED) near his home complaining of severe sore throat and difficulty swallowing. He also reported that he was never vaccinated against diphtheria. After tests for Group A streptococcal antigen and infectious mononucleosis were negative, he was given oral augmentin (amoxicillin and clavulanate potassium) and discharged.

Four days later, he returned to the ED, afebrile but with worsening symptoms: chills, sweating, stridor (sounds indicating obstruction in the larynx), labored breathing, swollen neck, wheezing, nausea, and vomiting. His arterial pO2 was diminished, and X-rays showed soft-tissue swelling, an enlarged epiglottis, and an opaque left lung. He was intubated and admitted to the intensive- care unit with a diagnosis of impending respiratory failure. Laryngoscopy produced yellow exudates from the tonsils, posterior pharynx, soft palate, and anterior pharyngeal folds. For the next four days, the patient was treated with azithromycin, ceftriaxone, nafcillin, and steroids. Nevertheless, he became hypotensive and febrile, and continued to fail. Culture results showed MRSA was present in the sputum, and a throat swab for C diphtheriae was negative.

After eight days, a chest X-ray showed infiltrates were present in both lungs. Atracheostomy produced white exudates consistent with C diphtheriae infection. A typical C diphtheriae pseudomembrane covered the epiglottis and associated structures. Gram stains revealed the presence of Gram-positive rods, Gram-positive cocci, and yeast. More antibiotics were given – penicillin, gentamicin, vancomycin – and a diphtheria antitoxin (DAT) was added. Specimens cultured from the pseudomembrane were negative, but a polymerase chain reaction (PCR) test performed at the CDC proved that C diphtheriae toxin genes were present. Despite 17 days of treatment, cardiac complications ensued, and the patient died. The state health department and the CDC were notified. All previous contacts, possibly exposed to his respiratory secretions, were tested by PCR and cultured for C diphtheriae according to the CDC protocol. All contacts were given prophylactic antibiotics and a diphtheria toxoid vaccine, unless a booster had been given within the last five years.2

Diphtheria – Diphtheria is a life-threatening disease caused by a toxin produced by C diphtheriae and rarely by C ulcerans or C pseudotuberculosis. The deadly toxin produced is responsible for the pseudomembrane formed in the throat when the respiratory system is attacked. Transmission is through respiratory droplets, contact with skin lesions, and consumption of raw milk and dairy products. Though sporadically reported in the United States, diphtheria is endemic in many developing countries from unvaccinated populations. Travelers who are not immunized or have not had updated booster vaccine are at extreme risk if they are exposed.2

Clinical diagnosis’. The disease symptoms are sore throat, low- grade fever, and difficulty swallowing. Membranous nasopharyngitis or obstructive laryngotracheitis with swelling of the neck are observed in severe cases. Skin ulcers and the complications of airway obstruction, myocarditis, polyneuritis, and acute tubular necrosis may also occur. The diagnosis of diphtheria is primarily clinical, and suspected cases must be reported immediately to the CDC. Treatment, screening by a microbiology lab, and confirmation by the CDC are paramount. The patient must be isolated with universal precautions observed.2,3

Laboratory diagnosis – Culture and identification: Multiple swab samples are taken from the nose, throat, or any membranes present and transported (e.g., in Aimes semisolid media) without delay to the microbiology lab for culture. Advance notice is necessary to assure that correct processing for C diphtheriae will be performed. State departments of health and the CDC require notification of a suspected case.

Primary plating is done on routine 5% sheep-blood agar (SBA) and at least one selective medium, such as Cystine Tellurite blood agar (CTBA) or fresh Tinsdale medium. Growth on SBA produces white or opaque colonies; on CTBA, black colonies appear, providing presumptive identification after 18 to 24 hours at 37[degrees]C in 5% CO2. Because other coryneform bacteria produce similar colonies (usually smaller), fresh Tinsdale medium is recommended to demonstrate both tellurite reductase (black colonies) and cystinase enzyme activity (brown halo around colonies). Also available is selective media made with 5% SBA and 100 mug of fosfomycin per mL plus 12.5 [mu]g of glucose-6-phosphate per mL that inhibits most other coryneforms. Disks containing 50 [mu]g of each compound (BD Diagnostics, Sparks, MD) can be used as an alternative. Gram stain of colonies reveals pleomorphic Gram-positive rods. Subculture to Loeffler or Pai slants for purity is required prior to biochemical testing. Methods of biochemical testing include von Graevenitz and Funke (the CDC Special Bacteriology Reference Lab); API (RAPID) Coryne System (bioMerieux, Marcy l’Etoile, France); and the RapID CB Plus system (Remel, Lenexa, KA).3,4

Susceptibility testing: Until recent interpretive data supplied by the Clinical and Laboratory Standards Institute (CLSI) became available, testing methods and susceptibility results created confusion to a laboratory isolating any Corynebacterium spp. CLSI recommends the broth-dilution method with media designed for such fastidious organisms as Campylobacter spp. and Helicobacter spp. (CAMHB-LHB) but not disk diffusion. Some researchers have used the E- test (AB Biodisk, Piscataway, NJ) successfully; however, because Cdiphtheriae continues to exhibit in vivo susceptibility to penicillin and – with some exception – to erythromycin and other macrolides, susceptibility testing has not had high priority.5

Toxin testing; The WHO Streptococcus and Diphtheria Reference Unit (SDRU) modified the Elek Test, which employs antitoxin- impregnated disks that create precipitin lines (a positive reaction) when toxin-producing C diphtheriae is present on an agar plate after 24 hours of incubation. This method was found useful to detect the diphtheria toxin in the 1990s epidemic in Russia and the Ukraine. More recently, the WHO SDRU has developed a three-hour enzyme- linked immunosorbant assay, which can be performed on a culture- grown isolate of the organism. At present, a real-time PCR fluorescence test can be performed directly (from a clinical specimen) to detect the A and B subunits of the tax gene in C diphtheriae or (C ulcerans or C pseudotuberculosis. PCR results require confirmation with culture, histopathology, or an epidemiologic source before diagnosis is considered complete.4

Epidemiologic testing: Originally, the three biotypes of C diphtheriae – gravis, intermedius, and mitis – were differentiated on the basis of size and colony morphology. Discovery of numerous other biotypes not as easily distinguished, however, makes complete laboratory identification necessary (refer to Culture and identification). Outbreaks in Russia and the Ukraine in the 1990s were coincident with improved epidemiologic methods that applied molecular techniques to the typing of C diphtheriae elsewhere in the world. Examples include whole-cell peptide analysis, whole-genome restriction fragment-length polymorphism or (RFLP), ribotyping, and the more recent spoligotyping system, similar to the oligonucleotide typing for M tuberculosis. Methods, such as clonal grouping, genetic spacer region information, and so forth are also useful in differentiating endemic strains from those imported from other countries.3,4 Therapy: Diphtheria antitoxin is available only from the CDC and should be given, without waiting for laboratory confirmation, when nasopharyngeal or laryngotracheal symptoms appear. Because the disease may not produce immunity during convalescence, a diphtheria-toxoid vaccine should be given as well.3 Antibiotics are effective against C diphtheriae bacteria but not against the toxin. Thus, they are administered to eradicate and prevent transmission of the bacteria. Patient contacts must be sputum cultured for presence of the organism, and they should receive prophylactic antibiotics to eliminate any bacteria that may be toxin producing. Penicillin and erythromycin are usually given along with diphtheria toxoid vaccine unless vaccination or a booster has been performed within five years. The CDC protocol for children to receive diphtheria-pertussis-tetanus vaccine (DTaP) should be followed with appropriate boosters from two months to 12 years, followed by boosters every 10 years thereafter. Information is available for travelers from state health departments and at www.cdc.gov.2,3,4

Epidemiology: Because vaccination has become part of U.S. children’s health-maintenance program, the incidence of diphtheria has not been a threat since the 1950s. The increase in foreign travel, however, particularly to endemic areas (e.g., Haiti), changed previous patterns of transmission. When information regarding a patient’s travel, vaccination history, and possible exposure to infection are not immediately available to the attending physician, the patient fatality rate is 5% to 10%. Otherwise, standard procedure in any ED is first to rule out the common infections of Group A streptococcus, infectious mononucleosis, or viral infection. These procedures were followed in the case presented here.2,3

Worldwide immunization with DTaP vaccine has also prevented outbreaks of infection with C diphtheriae since the 1950s, but the most widespread and devastating outbreak in history occurred in 1994 in former Soviet countries. In Tajikstan, there were 31.8 cases per 100,000; in Russia, 26.2 cases per 100,000 – rates nearly 30 times those in the United States. WHO workers discovered that a diphtheria epidemic of 14,000 cases originated during the Soviet/ Afghanistan war in the 1980s. Soldiers returning home carried with them the deadly toxin-producing C diphtheriae. While outbreaks reached 12,000 victims in 1991, only 60% of Russian children (

Another astounding discovery was that immunologists, trained in the former Soviet Union, were not allowed access to “Western medical journals.” Lack of expertise led to the erroneous opinion among physicians that giving vaccines to children or families who were ill (for any other reason) was dangerous. Even after the 1994 epidemic ended, pertussis and tetanus were not combined with diphtheria (as in the western DTaP) because of continuing unfounded fears, use of outdated vaccine, or the unmanageable cost to purchase, transport, and store effective vaccines.6

In 2001, the incidence of U.S. children (ages 19 to 35 months) who had the required three doses of diphtheria-toxoid vaccine was 95%. When all U.S. residents were tested for antibodies considered “protective,” however, the percentage in children ages six to 11 years with a protective titer was 91%, but declined to 30% in adults aged 60 to 69 years. These startling changes occurred because the recommended boosters were not given every 10 years (or five years for contact exposure). From 1980 to 2001, there were 53 sporadic cases of possible or confirmed cases of C diphtheriae reported to CDC. Genetic testing revealed clonal similarity among all isolates found in the United States and the former Soviet Union.2,3

CASE II: Vibrio cholene – After vacationing in Southeast Asia, a 21-year-old U.S. college student became ill and presented to the ED. He admitted eating fried rice and an iced drink from a street vendor the day before his flight back to the United States. Nausea and vomiting were followed by watery diarrhea, abdominal cramps, and the devastation resulting from 15 bowel movements per day. Examination showed the patient suffered dehydration and orthostatic blood- pressure changes. Noted among his blood-chemistry test results were electrolytes, potassium (K) 2.8 mEq/L (critical 6.0 mEq/ L), and pCO^sub 2^ 22 mm/Hg (critical 40 mm/Hg). Fortunately, the physician had prior knowledge of the patient’s travel history to an endemic region. That information led him to order a microbiology culture and to notify the lab to look for Vibrio spp. Thus, the selective agar for Vibrio spp, thiosulfate- citrate-bile salts (TCBS) agar, was included in the initial stool processing. Immediate therapy, followed with intravenous Ringer’s lactate (electrolyte) solution administered as “replacement therapy” until he was stabilized. The stool culture grew toxigenic Vibrio cholerae O1.7 [Note: Information regarding antibiotic therapy was not available.]

Cholera – The cholera enterotoxin produced by Vibrio cholerae stimulates adenyl cyclase, an intestinal enzyme that causes secretion of large volumes of watery fluid (as much as one L/hour). The loss electrolytes – sodium, bicarbonate, potassium – leads to rapid dehydration, shock, and death in 50% of its victims. Unless immediate treatment is provided, hypokalemia, metabolic acidosis followed by hypovolemic and shock, renal and circulatory failure, are likely. Without benefit of rehydration therapy given intravenously along with antibiotic therapy, a patient experiencing severe diarrhea may rapidly progress to dehydration and death.7,8

Although cholera is not spread from person to person, the ingestion of V cholerae O1 through contaminated shellfish, water, ice, rice, and other foods exposed to “brackish” water (warm, salty- fresh), is a common source of contamination. The causes of epidemic cholera disease usually involve the non-invasive, toxin-producing V cholerae O1 serogroup (biotypes “Classical” and “El Tor”) or the more recent O139 serogroup found in India and Bangladesh, 1994. The antigenic, epidemic, and clinical characteristics of these toxic strains are distinctively different from those of the non-toxigenic strain, non-O1 V cholerae. The non-O1 strains do not agglutinate O1 or 0139 antisera but have phenotypic similarities to the toxigenic strains. Clinically, the gastroenteritis infection is usually mild to moderate, but unlike their toxigenic counter-species, the non-O1 strains often cause septicemia, particularly in high-risk groups (e.g., patients with liver disease and malignancies); the fatality rate can be from 47% to 65%. Non-O1 strains are associated with smaller, less severe outbreaks related to ingestion of contaminated seafood.7,8,9,10

Laboratory diagnosis – Isolation and culture: Routine enteric microbiology does not include special media to isolate V cholerae from stool. The lab must be alerted to the possibility of patient exposure to ensure that isolation and biochemical processing, specific for Vibrio spp., will be performed. Without information regarding seafood consumption or wound exposure to brackish water, this crucial diagnosis can be missed. Unless inoculation of media occurs within two to four hours, specimens should be preserved in a transport medium (e.g., Cary-Blair) to preserve viability. Vibrio spp. will grow on routine sheep-blood agar and MacConkey agar (MAC), but they cannot be distinguished from other sucrose-fermenting intestinal flora that may be present. Selective media, such as TCBS agar or a chromogenic agar (CHROMagar Microbiology, Paris, France), can be employed to select out these pathogens from normal enteric organisms. Phenotypic characteristics of the species include Gram- negative; facultative-anaerobic; and straight, curved, or comma- shaped rods. Noted for motility when grown in liquid medium, they are described as “darting” or “twisting,” a result of their mono- or multitrichous flagella.7,9

Identification: Agglutination tests using O1 or O139 antisera can provide presumptive identification. Other methods such as direct fluorescent-antibody and latex-agglutination tests require experienced interpretation of results. Commercial identification systems, manual or automated, have poor reliability for speciation ranging from 50% to 96% for V cholerae. Because microbiology labs may not have personnel or appropriate supplies to detect members of the Vibrionaceae family, specimens in question are usually referred to public-health labs for complete identification. Examples of biochemical tests needed to screen members of the Vibrio spp. found in human specimens are shown in Table 1. Characteristic of many Vibrio spp., other than V cholerae, is the growth requirement of 1% NaCl, which must be added to all media and biochemicals. The unique motility feature and variable biochemical characteristics cannot be evaluated by the commercial/automated systems used by most clinical labs.9

Susceptibility testing: Antimicrobial testing, when clinically indicated, usually is performed by public-health laboratories where cases of suspected V cholerae have been referred for confirmation and toxin testing. Though not available for other Vibrio spp., CLSI guidelines are available for V cholerae and include testing for ampicillin, the tetracyclines, folate pathway inhibitors, and chloramphenicol. The strains – 01,0139, and non-O1, collectively, – are 90% susceptible in vitro to aminoglycosides, azithromycin, fluroquinolones, extended-spectrum cephalosporins, carbapenems, and monobactams. Resistance exists in certain strains (e.g., O1, El Tor, and O139 from India and Bangladesh) to the antibiotics sulfamethoxazole, trimethoprim, and chloramphenicol.9 Therapy: Treatment with oral rehydration salts (ORS, electrolyte solution) had proven success during cholera outbreaks dating back to World War II. But in recent years, the use of intravenous therapy and administration of antibiotics has helped decrease fatality rates in severe cases. Fluid loss and the duration of illness have been dramatically reduced by antimicrobial therapy. When mild or moderate disease is present, ORS without antibiotic therapy is usually sufficient. Use of normal saline – because it does not contain the necessary electrolytes – should never be used to treat cholera.7 To avoid selective resistance, antimicrobial treatment is administered only when clinically relevant and after lab confirmation. Therapy with ciprofloxacin and doxycycline (for adults) and erythromycin (for children) is effective. WHO guidelines confirm the possibility of selective resistance and suggest that indiscriminate use may be ineffective in some cases. There are no vaccines authorized for travelers to endemic areas. Genetic engineering may resolve this dilemma with the development of a probiotic, a bacterial agent capable of binding to and neutralizing the cholera toxin. Scientists engineered a harmless strain of Escherichia coli to mimic GMl host receptors that, in turn, interfere with the attachment of Vcholerae to the small intestine.” At present, the best protection includes avoiding drinking water that is not treated or boiled and ingesting raw or undercooked food (particularly seafood). Physician recognition of cholera symptoms and prompt adherence to recommended therapy are imperative to decrease mortality and morbidity.7

Epidemiology: Endemic in the Indian subcontinent (Bangladesh, India, and Pakistan), cholera has spread seven times in 185 years to involve the entire world. Prior to 1905, the Classical biotype of serogroup 01 was responsible for six pandemics. Biotype El Tor has spread from Asia to Africa and South America in the past 35 years. First seen in the 1990s in South Asia, serogroup O139 continues to be endemic there.9

In the former Soviet states in the late 1990s, sewage pipes leaked into pipes used for drinking water. The outcome was an onslaught of enteric pathogens that created some of the most devastating epidemics ever seen – typhoid, Shigella dysentery, cholera-diseases usually seen only in developing countries. Another disaster allowed Vibrio cholerae O1 to thrive in the brackish salty water created in southern Ukraine during a project to bring fresh water for irrigation from the north. The Dnieper River emptied into Saslyk Lake, a salty marsh and estuary of the Black Sea that offered a perfect ecologic niche for warm-saltwater-loving Vibrio spp. The result was not only an outbreak of cholera but also a breeding ground for malaria as well as West Nile and Sindbis viruses added to the mix as a result of the increased mosquito population.6

From 1995 to 1999, there were 51 U.S. cases of V cholerae O1 but none of O139. Because the safety of U.S. drinking water and sewage systems is rarely compromised, cases of cholera have been limited to victims who travel to endemic countries. Outbreaks have been sporadic, and related to foreign travel or consumption of Gulf Coast seafood.9 During the two major hurricanes in 2005 in Louisiana, water was compromised when levee damage and subsequent flooding destroyed sewage systems. Two cases of cholera caused by V cholerae O1, ensued after victims ingested contaminated seafood. Both were susceptible to all antibiotics tested. Pulse field gel electrophoresis confirmed that the isolates were identical. No epidemics were reported, and the patients were treated with ORS and ciprofloxacin. Despite one patient’s co-morbidities, the two returned to their previous states of health.10

CASE III: Mycobacterium tuberculosis – A 31 -year-old man from Atlanta, GA, was presumed to be infected with XDR-TB. Against the CDC’s advice, he traveled to Paris and Greece, then to Italy and Prague, before he returned to the United States via Montreal. A preliminary diagnosis was made four months earlier when a chest X- ray revealed a lung lesion consistent with TB, and his tuberculin skin test (TST) was positive. The patient demonstrated none of the usual TB symptoms: productive cough, fever, chills, night sweats, and weight loss. Thus, he was not considered “highly infectious.” After 18 days of incubation, a culture performed at the Georgia Department of Human Resources grew mycobacteria, from which an isolate was referred to the CDC for confirmation and susceptibility testing. The patient was treated and told the organism was probably the MDR-TB, not XDR-TB, as originally suspected. He was then transferred to the National Jewish Medical and Research Center in Denver for specialized treatment. There he was quarantined in a negative-pressure room, in which the air is decontaminated by ultraviolet light. He was isolated with universal precautions to prevent transmission of the highly resistant strain should there be any change in his infectious state. He had possibly compromised the health of some 80 airline passengers during his travels. Pending surgery to remove the lung lesion, treatment was begun with five of the second- and third-line antimycobacterial drugs: moxifloxacin, cycloserine, para-aminosalicylic acid or PAS, amikacin, clofazamine, thionamide, and linezolid. Later, physicians surgically removed the lung lesion and confirmed his isolate as MDR-TB, treatable with the fluoroquinolones, among other drugs. His antibiotic therapy will undoubtedly continue for two years.12,13,14

MDR-TB and XDR-TB – From 1993 to 1999, the U.S. National TB Surveillance System reported declining rates of both TB and MDR-TB, defined by its resistance to the first-line drugs, isoniazid (INH) and rifampin (RMP), and susceptible to second-line drugs. In October 2006, the WHO was forced to revise the MDR definition when a new strain appeared that was resistant to second- and even third-line drugs – the fluoroquinolones and the injectable drugs amikacin, capreomycin, and kanamycin. Infection with the XDR-TB strain leaves a paucity of treatment choices in the armamentarium of antibiotics. Spread like diphtheria through respiratory droplet nuclei, the transmission of TB bacilli from person to person can threaten many populations. From 2000 to 2006, researchers were encouraged when a decreasing number of HIV/TB-infected patients was noted. That number was soon offset by an increase in foreign-born cases, particularly Asian. The exact number of XDR-TB cases continues to be incomplete because of limited data from developing countries (e.g., Africa, India) where the strain predominates. Worldwide data for cases identified as TB and MDR-TB for 1993 through 2006 totaled 202,436. cases in the United States (only) identified as XDR-TB for 1993 through 2006 totaled 49.15

Diagnostic tests: In 2005, the CDC and the FDA approved a new test, known as the Quanti-FERON-TB Gold test (Cellestis Limited, Carnegie, Victoria, Australia), that detects both active and latent TB. Performed with an enzyme-linked immunosorbent assay technique, it measures the amount of interferon-gamma produced in a patient’s blood. Long recognized as the standard test for infection with M tuberculosis, the reliability of the TST has been contested. Crossreactivity with other mycobacteria, an increased positive reaction on repeat testing, and dependence for test results on an observer’s eye all are problematic. Though the newer test is not as subjective, controversy exists regarding which method has greater sensitivity and specificity when testing all populations: normal, HIV-infected, and other immune-compromised groups.16

Laboratory diagnosis – Specimen processing: Digestion and decontamination of both sputum and bronchoscopy specimens is accomplished first by liquefaction (to release mycobacteria into the mucin), then with sodium hydroxide (NaOH) to aid in removing bacterial contamination. Though not without limitations, the combined reagents, N-acetyl-L-cysteine (NALC), dithiothreitol and 2% NaOH, are agents commonly used for this purpose. Other contaminated specimens may require NaOH alone. Processing is required prior to planting a specimen on culture media (e.g., Lowenstein-Jensen or Middlebrook 7H10 agar) or transfer to a broth suitable for an automated detection system [e.g., BACTEC 12B broth used with the radiometric BACTEC 460TB system (BD Diagnostic Systems, Sparks, MD)].

Safety requirements, guidelines for equipment, and methods used, as well as recommendations for transport and storage of specimens in approved clinical labs, are published in the CDC and National Institutes of Health (NIH) instruction manual, Biosafety in Microbiological and Biomedical Laboratories.

Staining: Acid-fast stains are required to penetrate the mycolicacid residues present in the cell walls of mycobacteria. The fluorochrome-staining method, demonstrated with fluorescent microscopy, is preferred for high sensitivity and easier detection of acid-fast bacilli. Confirmation of positive smears continues to require the gold-standard Ziehl-Neelson smear. Together, these stains have a predictive value of >90% for M tuberculosis complex (MTBC) in sputum. They provide rapid screening for patients who may be highly infectious and require isolation precautions for the protection of others.16,17 Note: MTBC includes M tuberculosis, M bovis, M africanum, M microii, M canettii, M caprae, and M pinnipedii.

Culture and identification: Both solid and broth media are used to culture mycobacteria. A broth medium is used for rapid growth and suitable for automated detection and susceptibility-testing systems. Solid media, – Lowenstein-Jensen (egg-based), Middlebrook 7H10 or 7H11 (agar-based), and selective media containing antibiotics to deter overgrowth by non-mycobacteria – are considered the gold standard for confirmation and susceptibility testing. From slow- growing (seven to 14 days) solid media, the characteristics of growth, colony morphology, pigmentation, and optimal temperature requirements (35[degrees]C, 37[degrees]C, and 30[degrees]C) can be evaluated. Biochemical tests for speciation (e.g., niacin accumulation and nitrate reduction for M tuberculosis) can also be performed. For more rapid growth (

More recently, a rapid method of MTBC identification has been described by researchers at the Tuberculosis Research Laboratory, Beijing, China, as the PCR-reverse dot-blot hybridization assay. Results are available in only 2.5 hours after PCR processing. The procedure does not require an expensive sequencer and can be performed in clinical labs. Agreement with DNA sequencing was 99% for clinical strains compared to 90.6% for more conventional methods. Reference strains tested in the study had specificity and sensitivity of 100%.19

Strain typing; Epidemiologic studies require definitive information made possible by the development of molecular methods. Table 2 lists some of the methods commonly used for strain typing.18

Susceptibility testing – Agar proportion method: In the United States and Europe, the agar proportion method of susceptibility testing of slow-growing mycobacteria was initiated in the 1960s to become the gold standard for all antimycobacterial-drug testing, except pyrazinamide (PZA), which can be determined by automation. Middlebrook 7H10 agar is infused with specific concentrations of antibiotic agents (agar diffusion) or overlaid with disks impregnated with these drugs (disk elution). Both “low-” and “high- ” level susceptibility are tested. The proportion of resistant “mutants” and drug concentrations in the media is critical. A significant proportion of MTBC resistance, above which a drug may not be effective, is set at 1%.20

BACTEC 460TB: The BACTEC 460TB radiometric method continues as the automated system of choice, despite availability of several acceptable non-radiometric systems. Growth in BACTEC 12B broth and metabolism by mycobacteria of the [^sup 14^C] palmitic acid present in the broth produces ^sup 14^CO2 at a rate and amount proportional to the growth of mycobacteria present. Results are rapid – growth occurs in seven days compared to two to three weeks for the agar proportion method and both first- and second-line drugs can be tested. The percent of resistant mycobacteria cannot be estimated, and both false-positive and false-negative susceptibility can occur. The automated method is used as a “screen” until definitive results from the agar-proportion test are available. Confirmation of any new MDR- or XDR-TB strain requires results from the agar-proportion method.20

Therapy: The first-line drugs recommended by the CLSI for MTBC are INH, RMP, ethambutol (EMB), and PZA. INH, which acts mainly on the organism’s cell-wall mycolic-acid synthesis, is the drug of choice for latent or active TB infection. Its effectiveness against MTBC continued from its first introduction in 1952 until resistance appeared in the 1960s.20,21 The second-line drugs, (ethionamide, the aminoglycosides [e.g., amikacin, kanamycin] and the fluoroquinolones [e.g., moxifloxacin and levofloxacin]), are used when first-line drugs fail. They require a longer term of therapy, and are more toxic, less effective, and more expensive. Of primary concern, however, is the enhancement of opportunity for selective resistance.21,22

Two types of drug resistance are found in M tuberculosis’, primary – occurring in an untreated person – or, the more common, and acquired – emerging during therapy as a result of selective resistance. Typical of all bacteria, antibiotic resistance occurs in mycobacteria by a variety of mechanisms that include decreased uptake (e.g., dormant acid-fast bacilli), drug inactivation, (e.g., beta-lactamase production), increased efflux, (e.g., fluoroquinolone resistance), and alteration of the target site (e.g., INH and RMP).20

Epidemiology: Outbreaks of MDR-TB appeared in the United States in the 1980s and 1990s. Prompted to stop the increasing incidence, the CDC initiated the 1992 National Action Plan to Combat Resistant MDR-TB. The plan was instrumental in improving laboratory services with rapid and more accurate identification technology, more rapid susceptibility testing, improved infection control, and coordination with HIV test results. The result was a dramatic decrease in MDR-TB cases from 1993 to 1999 that correlated with an overall total U.S. decrease of 34% in total number of TB cases.15

After New York City’s MDR-TB epidemic in 1991 was attributed to inadequate antibiotic therapy in a group of homeless victims and AIDS patients, the first monitoring system, Directly Observed Therapy System, or DOTS, was instituted. The WHO had endorsed the method with a worldwide endeavor to monitor compliance. Unfortunately, former Soviet countries (e.g., Russia, Ukraine, and Belarus) that were medically uninformed and financially devastated, persisted in adhering to the outdated programs of the 1950s. Isolation in sanitariums (institutions for treatment of chronic diseases, e.g., TB) and treatment with one or two antibiotics (only part of worldwide therapy from 1980 to 1990) were still in place from the Soviet rule of Nikita Krushchev. Lack of funding was the primary factor that forced early discharge of patients and insufficient therapy – a perfect setup for selective resistance and the appearance of the MDR-TB strain. The cost of X-rays and surgical procedures to remove infected lungs and other organs, combined with the expense of antibiotics, made treating these patients prohibitive. Even the old methods of confining patients to sanatoriums and treating with one or two drugs became unmanageable with drug resistance spreading. They were simply left there to die. By 1998, the WHO noted that 25% of the former Soviet cases were multidrug resistant.6

At present, the greatest threat lies in developing countries where HIV/AIDS rampages out of control. According to a recent study in Natal, South Africa, co-infection with TB affects 80% of patients and mortality is nearly 40% per year despite therapy. The rate of 1.7% in MDR-TB cases (2000 through 2002) had increased to 9% (2003 through 2006) by the second survey. Analysis of 53 cases of XDR-TB showed only 50% had received therapy for TB. Within 16 days of the culture report, 52 of the 53 had died.23

In 2006, treatment guidelines for MDR-TB were prepared by the WHO, the American Thoracic Society (ATS), the CDC, and the Infectious Disease Society of America (IDSA). Further guidelines were written by the WHO in the Global Plan to Stop TB 2006-2015. Included are instructions for the proper management and administration of drugs in cases of MDR-TB and XDR-TB. The strategy and financial burden will require as much as $56 billion U.S. to cover all programs, (e.g., DOTS, the research and development of newer technology tools, and so forth).21,22

Summary – The cases presented here illustrate potential epidemic or pandemic events that once-silent pathogens portend. Developing countries, where defenses are limited, are primary targets. Of future concern are the developed countries that fail to use rigorous control measures established by the CDC, the WHO, and others to prevent the spread of infectious diseases. International travel has brought changes in demographics and a greater need for surveillance programs to control selective antibiotic resistance. In our first case, the patient’s death would likely have been avoided if he had adhered to the CDC-recornmended vaccine program. The fact remains that 20% to 60% of U.S. adults, including travelers to endemic areas, have not followed the diphtheria booster-vaccine schedule.2,3,4

Our second case demonstrates the ease of transmission through food and water that contribute to illness from cholera. The success of intravenous therapy or ORS treatment has reduced the number of deaths by approximately 3 million per year in Asia and Africa. Though development of oral vaccines may be promising, without water and sewage control, waterborne transmission of cholera is a continuing threat.7,8,10

The panic following our third case was a sample of the pandemonium an outbreak of XDR-TB would create. Lack of access to medical care and lack of funding in developing countries made selection of resistance from MDR- to XDR-TB a predictable event. The Global Plan to Stop TB 2006-2015, initiated to improve on the DOTS strategy to control TB/HTVand MDR-TB cases, targets 2015 for reducing overall prevalence and mortality from TB.21,22 Based on the principle that community healthcare is the best prevention, organizations that are dedicated to the control of emerging and re- emerging infection include the CDC, NIH, IDSA, ATS, and the National Academy of Science’s Institute of Medicine, to mention a few. Recently, they have established plans and programs to address problems of communication among scientists and to improve surveillance in the detection and monitoring of dangerous pathogens.1 The remarkable and ever-changing dynamics of microbial adaptation, however, requires enormous vigilance and financial priorities worldwide. A coordinated effort by both scientists and public-health leaders is needed if the onslaught of infectious threats is to be controlled.

Rapid nucleic-acid tests for identification

1. The AMTD kit (Gen-Probe, San Diego, CA) is adaptable for smear positive or smear negative specimens by targeting the 16S rRNA region of the genome to detect MTBC, but does not differentiate among species in the group.

2. For smear positive samples only and for species identification (M tuberculosis) the Amplicor system, (Roche Molecular Systems, Branchburg, NJ) targets the same 16S region.

3. The third method, BD Probe Tec strand displacement amplification (BD Diagnostic Systems, Sparks, MD) is an isothermal enzymatic process that uses IS6110 (MTBC) and the 16S rRNA gene of mycobacteria.17,18

Molecular Methods for Strain Typing MTBC

1. IS6110a – restriction fragment-length polymorphism, known as RFLP, was first used as a DNA probe to sequence MTBC strains. This insertion sequence is commonly found in these strains. A newer method, “mycobacterial interspersed repetitive units” (MIRU-VNTR) that can accurately cluster epidemiologically related strains is expected to replace the older IS6110 RFLP.

2. Spoligotyping-The “spacer oligotyping” method is based on a region of the genome that has non-repetitive short spacer sequences. PCR amplification aids in the specific identification of a group of oligonucleotides in the M tuberculosis genome. Not recommended for routine clinical laboratories, the method requires complex and multistep hybridization.

3. PCR strategies-Certain genetic regions “variable-number tandem repeats” (VNTR) that can be amplified and coded to correspond to the number of repeated units in each region.

4. Whole-genome fingerprinting- High-density oligonucleotide microarray testing: 20 probe pairs target the intergenetic region of M tuberculosis and can be used to analyze a small number of strains by detecting patterns of deletions. This method is expected to become commercially available for future clinical-laboratory use.18

References

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18. Wu X, Zhang J, Liang J, et al. Comparison of three methods for rapid identification of mycobacterial clinical isolates to the species level. J Clin Microbiol. 2007;45(6):1898-1903.

19. Woods GL, Warren NG, Inderlied CB. Susceptibility test methods: mycobacteria, Nocardia, and other actinomycetes. In: Murray PR, Jorgensen JH, Pfeller MA, et al. Manual of Clinical Microbiology. Vol 1. 9th ed. Washington, DC: ASM Press; 2007:1223- 1247.

20. Lettieri CJ. The emergence and impact of extensively drug- resistant tuberculosis. Medscape Today. http://www. medscape.com/ viewarticle/557459. Accessed May 8, 2008.

21. Migliroi GB, Loddenkemper R, Blasi F, Raviglione MC. 125 years after Robert Koch’s discovery of the tubercle bacillus: the new XDR-TB threat. Is “science” enough to tackle the epidemic? Eur Respir J. 2007;29:423-427. http://www.erj.ersjournals.com/cgi/ reprint/29/3/423.pdf. Accessed May 8, 2008.

22. Bartlett J. MDR and XDR tuberculosis literature: commentary by Dr. John G. Bartlett-April 2007. Medscape Today. http:// www.medscape.com/viewarticle/555306. Accessed May 8, 2008.

By Cynthia B. Schofield, MT(CAMT), MPH

Cynthia B. Schofield, MT(CAMT), MPH, is a microbiology technical supervisor (Ret.) from the VA San Diego Healthcare System in California.

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