College of American Pathologists
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In microbiology lab, five not-so-easy pieces

December 2002
Cover Story

William Check, PhD

When the call goes out to the clinical microbiology laboratory to "round up the usual suspects," Bartonella, Cyclospora, Ehrlichia, Mycoplasma, and Tropheryma are not typically the first names on people's minds. So why was a whole symposium devoted to these microbes at the 2001 Interscience Conference on Antimicrobial Agents and Chemotherapy?

"All these organisms fit into that unique category that we have lumped with Haemophilus in the past as fastidious organisms," explains co-convener David Welch, PhD, national associate director of microbiology at Laboratory Corp. of America, Dallas. "In the subtitle for the session we called them recondite," he says. "Recondite implies something hidden. That's appropriate—organisms like Mycoplasma and Bartonella are at the far end of the spectrum of fastidiousness and of culturability. On the other hand, they are not uncommon causes of clinical syndromes."

Others agree. "Most people think these are rare organisms, but they’re not if you know how to look for them," says David Walker, MD, professor and chairman of the Department of Pathology and director of the WHO Collaborating Center for Tropical Diseases at the University of Texas Medical Branch, Galveston.

"If someone asked me how to detect these organisms, I would have to go to a textbook," admits co-convener Bruce Hanna, PhD, associate professor of pathology and microbiology at the New York University School of Medicine and director of clinical microbiology at Bellevue Hospital. For this reason, the symposium focused on methods of detection. "These organisms are not easy to grow," Dr. Hanna says. "Antibodies can sometimes be confusing and difficult to interpret. And PCR is promising but still in progress."

Because of their occult nature, these five microbes have been associated with mysterious maladies and have given rise to mistaken identity, misattribution, and even an accusation of murder. Molecular methods are now illuminating their identities. Use of nucleic acid amplification assays for 16S rRNA sequences "has brought about a new approach to the identification of microbial pathogens that cannot be cultivated in the laboratory," wrote David Relman, MD, and Stanley Falkow, PhD, of Stanford University, who have worked on several such organisms. "These results suggest the existence of a far greater microbial diversity among human pathogens than has been so far appreciated with culture-dependent methods" (Infect Agents Dis. 1992;1:245).

"From my perspective directing a large referral laboratory that services the gamut of infectious diseases from community practice to a major teaching hospital, it is an exciting time to be in microbiology," says CAPMicrobiology Resource Committee member Deirdre Church, MD, PhD. Dr. Church is division head of microbiology in the Department of Pathology and Laboratory Medicine, Calgary (Alberta) Laboratory Services. "With molecular technology coming onboard, we can have a much better understanding of where a whole host of organisms fits into disease patterns." Calgary Laboratory Services tests for all of the microbes discussed except Ehrlichia, whose tick vector is not found in its region.

Nothing illustrates more clearly the confusion that an unidentified microbe can cause than what one review called the "long and circuitous history" of Bartonella. Bartonellosis was long thought to be a uniquely Peruvian illness called Carrion's disease, after Peruvian medical student Daniel Carrion. In 1885, Carrion inoculated himself with material from a child with a skin disease called verruga peruana in an attempt to discover the natural history of this illness. Surprisingly, Carrion became severely ill and died six weeks later. His symptoms were those of another, often-fatal Peruvian disease, Oroya fever. Carrion thus inadvertently proved that these illnesses are two phases of infection with the same organism, later determined to be Bartonella bacilliformis. (A colleague who assisted with Carrion's self-inoculation was briefly charged with homicide.)

Bartonellosis was synonymous with Carrion's disease for precisely one century. In 1985, a technologist named Diane Hensel working in a laboratory directed by Dr. Welch in Oklahoma City recovered and worked up isolates from blood cultures of several HIV-infected patients. Dr. Welch recalls that they saw one isolate of this "Campylobacter-like" organism each year for the next few years.

In 1990, they published a detailed report on two cases in the New England Journal of Medicine. In the same issue (vol. 323, no. 23) were articles on "the agent of bacillary angiomatosis [BA]" from Stanford University and a report on bacillary peliosis hepatis from the University of California, San Francisco. ("Peliosis" is a histological term for cysts that become filled with red blood cells.) Both Dr. Welch's paper and the one from Stanford suggested that the unknown bacillus was related to Rochalimaea quintana. Subsequent exchange of material among the laboratories and 16S rRNA analysis showed that the three groups were working with the same organism, which was first named Rochalimaea henselae (after Dr. Welch's technologist) and then Bartonella henselae, when sequencing revealed identity with that genus.

Soon after, polymerase chain reaction analysis showed the presence of Bartonella henselae in lymph nodes of children with cat-scratch disease, which had previously been misattributed to Afipia felis. "Fifty years after the clinical entity cat-scratch disease was described, we finally found the true cause," Dr. Welch says.

A third Bartonella species that causes human disease is B. quintana, the agent of trench fever. B. quintana has also been found to cause bacteremia in homeless people, possibly transmitted by body lice and often leading to severe endocarditis. Dr. Welch notes that B. henselae has also been found to cause endocarditis, typically in patients who have had cat exposure.

"B. henselae as a cause of opportunistic infections in HIV-infected persons is declining with the effective preventive regimens currently employed," he says. "Now we are increasingly seeing this pathogen as an agent of culture-negative endocarditis in non-immunocompromised persons, many of them elderly. The majority end up having cardiac valve replacement."

The most common reason to test for Bartonella is suspicion in a child of apparent cat-scratch disease, of which there are about 20,000 cases each year in the United States. It's "more common than Lyme disease," says Dr. Welch. In 10 percent of cases, "atypical" ocular or central nervous system complications occur. Bartonella may also cause culture-negative endocarditis.

For identification, Dr. Welch says, "there are not many direct detection methods worth pursuing." It is possible to visualize B. bacilliformis in blood samples and B. henselae in tissue or skin with Warthin-Starry silver stain.

"Cultures are not recommended, especially for cat-scratch disease," Dr. Welch says. "Efficiency of recovery is not good, even with the shell vial technique." Cultures may be done for other manifestations on blood- or chocolate-enriched medium with prolonged incubation (up to 14 days). Recovery of Bartonella spp in cultures is poor, partly because of broader dependence on continuous monitoring systems that hold samples for less than seven days and that are broth-based. "Bartonella does better on solid media," Dr. Welch says.

He calls serology "the mainstay" of diagnosis. Immunofluorescent antibody tests are 84 percent to 95 percent sensitive and 94 percent to 98 percent specific. More recently, an IgG/IgM EIA showed 85 percent sensitivity and >98 percent specificity in PCR-proven cases of cat-scratch disease (Clin Inf Dis. 2001;33:1852–1858). Dr. Welch cautions that cross-reactions occur with Coxiella burnetii and Chlamydia spp.

"Most laboratories are getting geared up to do molecular diagnostic testing," he says. "If you have a molecular section in your microbiology laboratory, it is certainly reasonable to consider PCR for Bartonella." PCR, he says, "is no more of a homebrew method than serology, since serology needs a source of antigen that is not commercially available." PCR-based methods for B. henselae and other Bartonella species have been published (Am J Clin Pathol. 2001;115:900–909). "As PCR methods become more refined, they become more practical for the general microbiology laboratory setting," Dr. Welch says.

While not all laboratories will set up testing for Bartonella, Dr. Welch says, "infections due to Bartonella are common enough that all laboratories should at least have a place to get serology done routinely and a place for getting PCR-based assays done if indicated." PCR can be helpful for suspected infective endocarditis when tissue is available, for CNS disease, and possibly when tissue is available from fine-needle aspirate of a lymph node where serology is negative or equivocal.

Whether typical cat-scratch disease needs treatment is "subject to debate," Dr. Welch says. If an antibiotic is used, azithromycin is the first choice. Other manifestations of B. henselae or B. quintana can be treated with erythromycin or doxycycline. Beta-lactams are largely ineffective. "In vitro tests don't predict clinical response," Dr. Welch says.

Dr. Church agrees that testing for Bartonella is indicated in cat-scratch disease with involved lymph nodes and for endocarditis with multiple negative cultures. "We have worked hard to find it in AIDS patients," she says. "But in retrospect, we are even more likely to see it in a child with a cat or kitten at home or in elderly or homeless people. It is probably a lot more prevalent than we think."

Bartonella can be retrieved from an automated blood culture system, in Dr. Church's experience. "One of the keys in blood cultures is prolonged incubation," she says. "If we suspect Bartonella, we incubate cultures for four weeks. If we get a positive signal, we try to retrieve an organism on plated media." Dr. Church confirms with molecular testing using an in-house PCR assay. She agrees that Bartonella can be discovered via histopathological examination of lymph node or heart valve using special stains or PCR. "We don't do serology," she says, "but we could send that to a reference laboratory. It takes a couple of weeks to get results."

The unmasking of Bartonella began in 1990, but the identification and clinical importance of the "enigmatic parasite" Cyclospora cayetanensis is even more recent, dating from 1993. In retrospect, three 1977–1978 cases in New Guinea were caused by this protozoan, but they were misattributed to a close relative, Isospora, says Charles R. Sterling, PhD, professor of veterinary science and microbiology at the University of Arizona, Tucson.

For some time Cyclospora was misidentified as an alga. "People were confused by its autofluorescent properties, which many algae have," Dr. Sterling explains. "Also, we could not make out discrete structures inside the oocyst. We see many things in the environment like that, including algae." In 1993, a student of Dr. Sterling's confirmed Cyclospora's identity as a coccidian parasite, like Cryptosporidium and Toxoplasma. "The student was able to put the organism into culture and to develop infected phases," Dr. Sterling says. The trick was to incubate samples for two weeks at room temperature, inducing sporulation into oocysts. (Typical coccidian parasites sporulate in 24 hours.) After lysis with bile salts and trypsin, "out popped two sporocysts with two sporozoites from each sporocyst, characterizing this as Cyclospora," Dr. Sterling says. The organism is not yet cultivable.

Waterborne outbreaks of Cyclospora have occurred in Nepal and Guatemala. Multiple food source outbreaks occurred from 1995 to 1999 in North America from imported Guatemalan and Mexican raspberries, basil, and leafy vegetables. Sequencing methods can now link patients within an outbreak to each other and to the implicated food. They can also link the outbreak organism to organisms in humans working in the field where the raspberries were picked.

The FDA now blocks the importation of Guatemalan raspberries during the spring and monitors compliance with preventive measures. No outbreaks have been associated with Guatemalan raspberries in the past two years.

Additional insight—and a new puzzle—arose from work that Dr. Sterling's group did at a Lima clinic.

"We obtained permission to do endoscopy on patients infected with Cyclospora," Dr. Sterling says. "This allowed us to see where the parasite develops in the intestinal tract." Jejunal biopsies revealed intestinal stages, implying that the parasite completes its life cycle within the human host. The parasite causes intestinal inflammation, which produces the prolonged watery diarrhea characteristic of this illness. However, Dr. Sterling says, "We may see only a few organisms, yet on biopsy see profound inflammation. How is it caused? No one has isolated toxins from this group of organisms."

The parasite's development is a second puzzle. When shed in feces, it is in a nonsporulated phase. But organisms found on fruit are already sporulated. How and where do oocysts arise? Where do they go? One "guess," Dr. Sterling says, is that they are dormant for a portion of the year. "We can put Cryptosporidium in the refrigerator for 18 months and still some organisms retain infectivity," he says. Perhaps oocysts get on fruit via an environmental source. "One suggestion," Dr. Sterling says, "is that insecticide solutions are made up with contaminated water."

He calls Cyclospora symptoms "classic" for enteric disease: Profuse watery diarrhea (which may be intermittent for up to 40 days), with bloating and flatulence, is seen in nearly 100 percent of patients, with weight loss and nausea in 80 percent.

For diagnosis, an acid-fast stain—found in many laboratories because it is also used for Cryptosporidium—is used. However, Cyclospora oocysts are acid-fast variable; some don't pick up the stain at all. A modified safranin stain, which is heated slightly in a microwave, was developed at the Centers for Disease Control and Prevention. "It is important to have a good ocular micrometer," Dr. Sterling adds, "since Cyclospora oocysts are eight to 10 micrometers in diameter, whereas Cryptosporidium oocysts are rarely more than six microns in diameter."

These are "relatively nonspecific techniques," Dr. Sterling acknowledges, "yet that's what we use in the laboratory today." PCR is more specific, but it hasn't found widespread application. "Everyone agrees we need better primers before we have robust PCR testing for this organism," Dr. Sterling says.

When Cyclospora infection is recognized, treatment with trimethoprim/sulfamethoxazole for five to seven days is more effective than ciprofloxacin, he says.

In the laboratory, Dr. Church says, the main diagnostic test remains a stool sample for O&P examination and special stains, such as modified Kinyoun acid fast, on concentrated stool specimens. Cyclospora is structurally unlike any other parasite in stool except for Cryptosporidium, she notes. Dr. Church would not do a special stain on a stool specimen routinely; she would do it only if the patient had a suspicious history. "These are patients who come to a consultant, usually a gastroenterologist, with unresolved disease," she says.

The story of human ehrlichioses has unfolded in three chapters over the past 15 years, with an indispensable contribution from molecular methods. Dr. Walker defines an Ehrlichia as a "small, obligately intracellular, gram-negative bacterium with characteristic dimorphic appearance and cell wall ultrastructure that resides in cytoplasmic endosomes." In 1986, human patients were first observed with mononuclear cells containing ehrlichia-like organisms. In the second chapter, Dr. Walker and others described in 1994 six patients with neutrophils or granulocytes containing inclusions suggesting ehrlichial infection (J Clin Microbiol. 1994;32:589–595).

Human monocytic ehrlichiosis (HME) and human granulocytic ehrlichiosis (HGE) have similar nonspecific presentations, with nearly ubiquitous fever and malaise and less frequent nausea, cough, and confusion. Arthralgias may occur. Leukopenia, lymphocytopenia, and thrombocytopenia are present, and elevated liver enzymes are common. "Ehrlichioses can be life-threatening," Dr. Walker says. Adult respiratory distress syndrome and meningitis "are definitely not rare," he adds.

Analysis of 16S rRNA genes from the HME agent showed that it was a new species, named Ehrlichia chaffeensis after the town in Arkansas where the isolate originated. When the HGE agent was found, Dr. Walker and his colleagues performed a serological and PCR analysis comparing it to E. chaffeensis and two nonpathogenic strains, E. phagocytophila and E. equi. Results showed that the HGE agent differed from E. chaffeensis and was identical to the other two species. A more detailed evolutionary analysis published in 2001 by Dr. Walker‘s former student, J. Stephen Dumler, MD, of Johns Hopkins Medical Institutions, and coworkers resulted in a major reorganization of this group of organisms, as well as a new name for the HGE agent—Anaplasma phagocytophilum.

HME is transmitted by the lone star tick Amblyomma americanum; HGEis transmitted by Ixodes ticks. In true evolutionary fashion, these two microbes seem to have found complementary geographic niches. HME occurs primarily in the southeastern and south central regions of the United States, with an incidence in rural Missouri of 11 to 100 cases per 100,000 people. HGE's distribution is similar to that of Lyme disease (not surprising, since they share a vector): more prevalent in the upper midwest and northeast U.S., with an incidence in Connecticut of 51 cases per 100,000 people. HGE complicates workup of Lyme disease. The Deer-Associated Infection Study Group recently published an approach to differentiating Lyme disease, HGE, and babesiosis (Clin Infect Dis. 2002; 34: 1184– 1191). HGE also occurs widely in Europe.

Most recently, in chapter three of the human ehrlichiosis saga, human disease due to Ehrlichia ewingii has been reported in a few patients in Missouri, Oklahoma, and Tennessee, about 70 percent of whom were immunocompromised because of HIV infection, transplantation, or therapy for Crohn's disease.

Still under investigation are reports from Perm, Russia of patients possibly infected with an E. muris-like organism.

To avoid the difficulty that has attended diagnosis of Lyme disease, those working on human ehrlichioses formed the Consensus Approach for Ehrlichiosis (CAFE) Society. CAFE has met for several years with the goal of establishing criteria for diagnosing ehrlichioses, as well as sharing samples in a de facto proficiency program. "There has not been a big change in the last couple of meetings," Dr. Walker says, "which speaks to the slowness of getting recommended serology tests to market."
Diagnostic criteria for the ehrlichioses are "still emerging," Dr. Walker says. He lists several "arbitrary criteria" for diagnosing HME:

  • Seroconversion—a fourfold increase in titer or an anti-E. chaffeensis IFA >256.
  • Isolation of E. chaffeensis.
  • PCR detection of E. chaffeensis for two target genes.
  • Positive serology (IFA >64).
  • Identification of morulae on peripheral smear inside cells.
Candidate criteria for HGE are similar.

None of these criteria are ideal. "IFA is somewhat subjective," Dr. Walker says. Moreover, there is about 15 percent serological cross-reactivity between E. chaffeensis and Anaplasma phagocytophilum.

"PCR is a dangerous tool in the hands of some people because of contamination," he says. "Probing for two genes is desirable because you are less likely to screw up twice."Even with positive PCR, he says, "we like to have some additional confirmation, such as seeing morulae or a single supportive titer of 64 or greater.

"We all would like to see a test that is more precise and could be automated," Dr. Walker continues. "A few companies are interested, but right now this disease has not got priority. People think of it as a rare disease and as boutique testing, but in my opinion that is not correct. These diseases are quite common; they are just difficult to diagnose." Without good diagnostic methods, however, establishing their true incidence is difficult.

In the absence of definitive diagnostic criteria, Dr. Walker says, "I am definitely an advocate of treating patients empirically." The preferred agent is doxycycline, given for more than one week. Dr. Walker calls chloramphenicol "controversial," saying, "It probably doesn't work." Nor do quinolones or macrolides.

The first direct evidence for a bacterial etiology of Whipple's disease did not appear until 1991, when PCR was used to identify a partial 16S rRNA gene sequence. In 1992 Drs. Relman and Falkow confirmed this work, and the organism was recognized as an actinomycete and assigned the name Tropheryma whippelii. A highly accurate PCR test for diagnosis and monitoring was described in 1997 (Ann Intern Med. 1236: 520–527).

Didier Raoult, MD, professor of clinical microbiology and director of the rickettsial unit at the University of the Mediterranean in Marseilles, reported in 2000 the first cultivation of T. whippelii, from a 42-year-old man with blood culture-negative endocarditis (N Engl J Med. 342: 620–625). In the following year, Dr. Raoult isolated a second strain of T. whippelii from an intestinal biopsy of a patient with typical relapsing Whipple's disease. Almost 95 years after this condition was first described, the etiologic agent was reliably cultivable.

Still unanswere is whether there are carriers for T. whippelii, or whether it rarely occurs in intestinal mucosa that lacks histopathologic evidence of Whipple's disease.

This year, Dr. Raoult reported immunohistochemical detection of T. whippelii in lymph nodes. "Fifteen percent of circulating monocytes were positive to antibodies," he says. "This will be a good test. There is no serological test."

Right now PCR testing is too sensitive and nonspecific, Dr. Raoult says. "There is a lot of confusion in PCR diagnosis," he says. "Some researchers have reported PCR positivity in saliva of up to 30 percent of patients without Whipple's disease. At this time it is difficult to make the diagnosis on the basis of PCR alone." Also, antibiotics other than TMP/SMX are being evaluated.

"Tropheryma whippelii is an interesting organism," says Dr. Church. "We don't see it commonly. It manifests mostly in middle-aged white males who are otherwise well and who present with severe weight loss, diarrhea, and steatorrhea.

"One thing that has hampered our understanding of this organism and our ability to devise good tests is that we couldn't cultivate it until recently," Dr. Church continues. "Now it can be cultured in special systems, which should lead to genetic sequences to target." Dr. Church relates that a case of culture-negative endocarditis was recently diagnosed in Toronto in which T. whippelii was identified in heart valve tissue using the universal primer approach followed by PCR with specific primers. "The clinical laboratory only did the molecular testing because the histology of the cardiac tissue was highly suggestive of Tropheryma whippelii," she says. To eradicate the pathogen, the valve must be excised and replaced, and the patient must take antibiotics for weeks or months.

Mycoplasmas are "the smallest of the small" and "the simplest" of microbes, says George Kenny, PhD, professor of pathobiology in the School of Public Health and Community Medicine at the University of Washington, who has studied these organisms since the 1960s. Mycoplasmas are "extraordinarily difficult to culture," he says. "They have almost no synthetic abilities, so they require complex media, but there is no quality control of these media." Dr. Kenny discovered when he entered this field that it didn't matter whether commercial media supported the growth of established cultures; he needed to test it on patient isolates, since wild strains are harder to grow than cultivated ones.

Mycoplasma pneumoniae, the prime mycoplasmal pathogen, causes atypical, relatively mild "walking pneumonia." "Patients can be quite sick, with lung infiltrates, but still up and around, but feeling miserable," Dr. Kenny says. In a long-term (1963–1975) study of patients hospitalized with M. pneumoniae pneumonia, Dr. Kenny and his colleagues found that most eventually recovered. Many associations of M. pneumoniae with neurological disease have been reported. "Now that we have PCR, we should be able to settle these claims," he says.

In 1981, M. genitalium was first identified. That isolate's genome was sequenced and a PCR assay developed, which was used to implicate the organism in nongonococcal urethritis (NGU). There were no further isolates until 1996, when Danish workers developed a method to grow M. genitalium in tissue culture. The genome of M. genitalium appears to be a cut-down M. pneumoniae genome; until recently it was the smallest bacterial genome known, making it a subject for evolution research.

M. hominis causes mild systemic infections and possibly postpartum infections in women with ruptured membranes. Ureaplasma urealyticum was thought to be associated with NGU. "Now we are quite certain this is not so," Dr. Kenny says, "since 60 percent of healthy women carry it." He notes that U. urealyticum could still be important in postpartum fever and when found outside the urethra or vagina.

Dr. Kenny and his colleagues compared the rates of recovery of M. genitalium and U. urealyticum from urethral swabs of heterosexual men with NGU. M. genitalium was found in 22 percent of patients and four percent of controls, similar to incidences of Chlamydia trachomatis, showing that M. genitalium might play a major role in NGU. U. urealyticum, on the other hand, was recovered from 42 percent of patients and 50 percent of controls. "So perhaps we can get rid of the idea that we need to test for ureaplasmas in NGU," Dr. Kenny concludes (J Infect Dis. 2001;183: 269–275).

M. fermentans was claimed to be a co-pathogen in AIDS by HIV co-discoverer Luc Montanier, MD, of the Institut Pasteur, but this has been disproved. "It is commonly found in the genital tract and bloodstream of healthy people," Dr. Kenny says.

M. pneumoniae is readily culturable. In patients with X-ray-proven pneumonia and significant antibodies to this microbe, isolation had sensitivity of 64 percent and specificity of 88 percent, which meets the clinical laboratory's need. "Many bacteria have about that isolation rate," Dr. Kenny says. "You never get 100 percent—that's folly." Unfortunately, culture is not practical for diagnosis, since it may take several weeks. "We could only do this study because we could prove positivity by serodiagnosis," Dr. Kenny adds.

M. genitalium is "essentially unculturable in the clinical laboratory," he says. M. hominis requires a specialized medium but grows in two to five days, as does U. urealyticum.

Available EIA kits for M. pneumoniae measure IgM and IgG, since not everyone produces both types of antibody (J Clin Microbiol. 1992; 30: 1198–1204). "These assays are reasonably successful,"; Dr. Kenny says, "but I believe PCR would be more successful." Identification following PCR still requires Southern blot analysis, however, which is too time-consuming for routine laboratory diagnosis. "I am sure it will be reduced to a kit with a different indicator than Southern blot," Dr. Kenny says. "We have made our own." Japanese workers have described a real-time PCR assay (J Clin Microbiol. 2002;40: 3854– 3856).

Mycoplasmas have no cell wall, so they are not susceptible to beta-lactam antibiotics. They are susceptible to tetracyclines, quinolones, streptogramins, and aminglycosides. M. pneumoniae and M. genitalium are highly susceptible to macrolides, including erythromycin, but M. hominis is not. Acquired resistance to tetracycline, quinolones, and macrolides has been seen.

Dr. Church calls mycoplasmas "a confused area. M. pneumoniae is the most important one for us," she says, "since it is the most common atypical cause of community-acquired pneumonia." Of the "genital mycoplasmas"—M. genitalium, M. hominis, and U. urealyticum—she says, "Their role in clinical disease is not always clear."

Culture is not feasible for diagnosis, Dr. Church agrees. She finds serology not sensitive enough and nonspecific. "The best way to detect M. pneumoniae is with PCR," Dr. Church says. "There are a number of good in-house assays described, even a multiplex assay with Legionella pneumophila and Chlamydia pneumonia—the three major causes of atypical community-acquired pneumonia." Dr. Church's colleagues have developed such a multiplex assay setup in-house.

Of other mycoplasmas, she says, "Most laboratories are not too keen to spend a lot of time on the normal flora of the genital tract, especially Ureaplasma in vaginal specimens. So we need to look in defined clinical conditions." She refers cases of suspected infections with these organisms to the public health laboratory, which does culture.

In the near future, Dr. Church sees molecular technology as providing broader value than just illuminating the shadows around recondite microbes.

"Microbiology, particularly bacteriology, has been a heavily culture-based discipline," she says. "Now as we adopt testing with molecular technology, we are not just going to find new conditions." Even in common conditions like outpatient urinary tract infection and chronic prostatitis, molecular methods are likely to redefine the epidemiology and microbiology of those diseases.

With their culture-based techniques, microbiologists probably are not retrieving all of the organisms causing those syndromes, she says. "It is going to be a stretch for smaller laboratories to keep up," Dr. Church acknowledges.

"On behalf of their patients, they will have to be partnered with a larger laboratory, though it won’t necessarily be a reference laboratory," she says. "Molecular diagnosis will become common in many clinical laboratories in various regions over the next 10 years."

William Check is a medical writer in Wilmette, Ill.