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New understandings of mycoplasmal infections and disease
Clinical Microbiology Newsktter Vol. 17, No. 22
,
New Understandings Shyh-Ching Lo, M.D., Ph.D. Chief, Molecular Pathobiology n_-_-.-._._. “J _ZI._l__r.‘ _.._am4 -..1 l-arawuc n_.._^:.:_ “eparwnem 1nJ”“‘“u-y . Disease Pathology Armed Forces Institute of Pathology Washington, D.C. 203066000
Mycoplasmas, a heterogeneous group of the smallest organisms capabie of seif-replication, beiong to the class Mollicutes. More than 100 different species of mycoplasmas have been identified. Like other bacteria, each species has distinct biological and biochemical properties associated with its ability to produce disease. The genetic material has the characteristics typical #.Fp‘ ...-tn...,,5*~” .-._..#.-a W&G o&n 1J :o “#llJ ,-..xl.. “1 “fial~“IGJ. Tlr,3 111G~Wlr”rrrG about 10 to 15% of that of usual bacteria, and genes necessary for cell-wall synthesis are absent. The wall-free organisms are highly pleomorphic in shape and are nutritionally fastidious, requiring rich, complex media to support their growth in culture. Specifically, all mycoplasmas need cholesterol, which is normally added as an animal serum supplement to the culture medium. Some Mollicutes, the Ureaplasma spp., can use urea as an energy source. Mycoplasmas are medically important bacteria res_ponsiblefor a wide range of human diseases. The infections they produce are commonly misdiagnosed as viral infections (1,2) because of the organisms’ small cell size, tilterable property, and the absence of acute inflammation that is atypical in bacterial infections. Mycoplasmas
in Animal Diseases
Mycoplasmal and ureaplasmal infecCzMNEEJ 17(22)x9-176.1995
November 15,1995
of Mycoplasmal
Infections and Disease
tions in animals are of historic importance in the field of mycoplasmology. The organisms have iong been recognized as pathogens causing significant health problems in commercial and laboratory animals. About a century ago, Mycoplasma mycoides was first described as the infectious agent causing ~~~r.Y~:,..~L-..:.., ,,..,..-,..:a 1‘a cvlllitgluus uu”lllG,1^.__ yKXl”yrrcur*r”‘ (CBPP) in cattle. Subsequently, mycoplasmas and ureaplasmas have been isolated from essentially every domestic and laboratory animal. Infections are often associated with the development of disease of the respiratory and urogenital tracts, joints, and other tissues I?). Mvconlasmaa significant im\_,_ -.-, __~__‘___ have ..-. _ _a _.~ .._ ___.._ -... pact on agricultural economy and biomedical research. In mycoplasmology, great emphasis has been and continues to be placed on the patbogenesis, diagnosis, and treatment of mycoplasma1 infections and diseases in animals. These wall-less prokaryotes also infect and cause disease in plants and insects (4). Ifis not clear whether the microorganisms can be transmitted to animals by infected insects with subsequent disease production.
Although mycoplasmas cause a variety of acute, fulminant, and chronic debilitating disease in animals, they are responsible for only a few significant human illnesses such as respiratory and urogenital tract infections (5). In fact, the most common community-acquired pneumonia is caused by Mycoplasma pneumoniae. The previously documented human mycoplasElsevier
ma1 diseases are briefly summarized here. M. pneutnoniae infection
This organism causes pneumonia in approximately 2 million people each year (6). The most prominent histopathological change associated with infection is interstitial pneumonitis with. edema and significant thickening of the septa. Unlike most bacterial pneumonias, leukocytosis and increased immature neutrophil (“band”) counts are generally not prominent in the atypical form of pneumonia. The main clinical symptoms at onset of M. pneumoniae infections are fever, malaise, and head-
In This Issue New Understandings of Mycoplasmal Infections and Disease.. . . . . . . . . . . . . . . . . . . . . .169 New diagnostic methods have given us a greater understanding of the scope of mycoplasma infections in healthy and immunocompromisedpeople
Regulatory Issues Affecting the Diagnostic Laboratory.. . . . . :. . ,174 The impact of increased federal regulation on the clinical laboratory and its personnel
Mycobacterium genavetzse and Mycobacteriuttt aviutn Mixed Infection in an AIDS Patient.. . . . . . 175 A case report 01%-4399/95/$0.00+ 09.sd
ache. The disease syndromes include pneumonia, tracheobronchitis, and pharyngitis. Many extrapulmonary infections, including synovitis, myocarditis, pericarditis, and a variety of central nervous system syndromes are also commonly described (6). It is not completely clear whether these complications are due to direct involvement of mycoplasmas in the diseased organ systems or are produced by immunologically mediated reactions. Mycoplusma hominis and Ureaplasma ureulyticum infections
Both-organisms colonize the lower urogenital tract of many healthy individuals; however, they have been implicated as the cause of pelvic inflammatory disease (7,8). M. hominis can be isolated in culture from 2 to 10% of healthy persons and from more than 60% of women with abnormal vaginal discharge. Septicemia due to M. hominis is responsible for postpartum fever or postabortion fever in women (9). M. hominis infections at many~extragenital sites have been reported including joints, respiratory tract, and central nervous system. Low birth weight (less than 1,000 g) infants infected with U. urealyticum have a significantly higher frequency of developing respiratory distress syndrome (lo), but it is less clear if women colonized with U. urealyticum in their urogenital tracts are at higher risk of giving birth to premature or low birthweight babies (11). Mycoplasma genitalium
infection
This highly fastidious mycoplasma has been recovered from the urogenital (12) and respiratory tracts (13) and has been implicated as a potential cause of pneumonia and nongonococcal urethritis (NGU). Because this mycoplasma is essentially uncultivable, it has been difficult to assess the scope of infection it
produces without a good seroepidemiological assay. PCR is currently the only diagnostic technique available. In a recent study by PCR, M. genital& was associated with NGU (14). Interest in M. genitalium increased after Montagnier and his associates (15) detected the organism by 'PCRassay in blood samples of patients with AIDS. A serological assay recently developed by us (see below) also detected a high frequency of M. genitalium infection in intravenous drug users and male homosexuals with or without HIV infection (16). However, few patients with hemophilia and AIDS had serologic evidence of M. genitalium infection. Most importantly, M. genital& was a rather common infection (>40%) among nonHIV-infected patients attending clinics for treatment of sexually transmitted diseases. Our study showed that the organism is the cause of a current epidemic of sexually transmitted disease that has not been previously detected. The organism is a likely candidate to produce systemic infections in the immunocompromised AIDS population.
Mycoplasmas in AIDS Recent studies in our laboratories and others have revealed that patients at high risk for developing AIDS have unusually high frequencies of infections by certain Mycoplasma spp. such as M. fermentans and M. penetrans (16-19). Infections with these mycoplasmas might play an important role in certain disease processes in AIDS patients (19, 20). M. fermentans, M. penetrans, and M. pirum have been identified much more frequently in patients infected with HIV-l, with or without AIDS, than in non-HIV-infected control subjects. Mycoplasma fermentans Our laboratory (21) and later Hawkins
and colleagues (22) reported that this organism is found most ~~_mm~nlv -, in --- the ---urogenital tracts or urine of patients at high risk for AIDS. Systemic infection with M. fermentans in patients__yith AIDS is also common and can be detected by PCR assays of urine, blood, tissue, and other clinical specimens. Importantly, infection appears to be associated directly with functional deficits of the infected organs’or tissues. For example, M. fermentans infection of the kidney is associated with the development of nephropathy in AIDS patients (19). Recently, an HIV-seropositive patient, who was not immunosuppressed, developed focal glomerulosclerosis, and M. fermentans DNA was found in the kidney biopsy by using PCR. M. fermentans was detected 15 mo later in peripheral blood lymphocytes, urine, and throat specimens. By 3 mo later, the CD4+ lymphocyte count had fallen and the patient developed Pneumocystis carinii pneumonia (20). This finding strengthened the association between M. fermentans and the development of AIDS nephropathy and has illustrated that _M.J’ fprmontnnr .v.ay . .... ..._..” infwtigfi ..._.,“l. precede rapid progression to AIDS. Mycoplasmas can markedly enhance cytocidal effects of HIV-l on human CD4+ lymphocytes in vitro (23.24). The organism is also associated with an acute form of adult respiratory distress syndrome that may produce systemic manifestations in persons who apparently were previously healthy (25,26). These observations suggest that M. fermentans may cause a heretofore unrecognized systemic form of human . mycoplasmal disease that might lead to a fatal outcome in nonimmunocompromised patients. Mycoplusma penetrans
In a study in our laboratory of urine samples obtained from 113 HIV-infected patients, this previously un--
NOTE: No responsibility is assumed by the Publisher for any injury and/or damage to persons or prop&y as a matter of products liability, negligence or otherwise, or from any use or ’ F nr ‘et “r pm&c&cc!4 be crti& au: *:n!e§s, i.? the ,z%ic:‘s pdgmciz, 5 iis:: i op.mtmo of zmy motto& p_md&, ms!r!zc!!oL _. i,ira. .I_ c-nn+s;n.=A __.._-. “I in .. . thr _.” mdr.4.l . ..” ._..... hrrrin . .. . . .. . . Nn .._ qpg+_l ” justiticd. Because of rapid advancea in medical sciences, we ncommcnd that the independent verification of diagnoses and drug dosages should be made. Discussions, views, and recommendations IWto medical procedures, choice of drugs, and dmg dosages are the responsibility of the authors. ClinicalMicrobiology Npwslener (ISSN 1069-417X) is issued twice monthly in one indexed volume per year by Elsevier Science Inc., 655 Avenue of the Americas, New York, NY 10010. Subscription price per year: $175.00 including postage and handling in the United States, Canada, and Mexico. Add $59.00 for postage in the rest of the world. Second-class postage paid at New York, NY and at additional mailing offkea. Postmastec Send address changes to Clinkol Microbiology Newsletter, Elsevier Science Inc., 655 Avenue of tbe Americas, New York, NY 1cKllO.
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Clinical Micmbiology Newsletter 17:22,1995
known human mycoplasma (27) was isolated more than 12 times from 6 patients. In a parallel study, it was not isolated from 98 HIV-negative, age-matched, healthy control individuals. The organism possesses a unique terminal structure by which it attaches to and penetrates eukaryotic cells. Extensive invasion of infected cells by M. penetrans produces cytopathic effects and may lead to cell death. By electron microscopic studies of urine sediments that grew M. penetrans in high titer, we determined that the organism, with its specialized tip structure, apparently adheres to the surface of and invades the cytoplasm of uroepithelial cells in the patient’s urogenital tract (28). Preliminary serologic studies using EIA and Western blot (immunoblot) analyses revealed a high prevalence (40%) of antibodies to M. penetrans in AIDS patients (29). In comparison, a low incidence (0.3%) of antibody is found in patients attending sexually transmitted disease clinics. In addition, of more than 150 HIV-negative patients with different non-AIDS-related disease states, including immune dysfunction and/or low leukocyte counts, none tested positive for M. penetrans antihodies. This newly discovered mycoplasma apparently is not a commensal organism in humans or just a simple opportunist commonly infecting immunocompromised hosts. It is uniquely associated with HIV- 1 infection and/or groups at high risk for AIDS (29). Subsequent serologic assays have revealed that the organism infects primarily male homosexuals with or without HIV infection (16). Few HIV-infected patients in other groups at high risk of AIDS are infected by this organism. Since Kaposi’s sarcoma occurs mainly in male homosexuals and not in other AIDS patients, a significant association can be made between serologic evidence of M. penefruns infection and the development of Kaposi’s sarcoma (p
Cliiicd
Microbiology
Newsletter 17:22,1995
from rectal swabs of patients with AIDS, including those patients known to be urine culture-positive or seropositive for M. penetruns, were not successful. Mycopla.sma pirutn
This organism also has an organized terminal structure and metabolizes both glucose and arginine for growth. All isolates of M. pirum have been recovered from human cell cultures. The true origin or natural host for this species has not been well-documented. Reports from Montagnier and his associates (15) of the isolation of M. pirum from primary cultures of peripheral blood mononuclear cells from AIDS patients suggested that the organism is of human origin. The mycoplasma apparently has been isolated on two occasions from the blood of patients with AIDS. However, by using ELISA to examine for specific antibodies to M. pirum lipid-associated membrane proteins, we found few patients with AIDS or healthy non-HIV-infected individuals with serologic evidence of M. pirum infection (29). The significance of M. pirum in AIDS is still unclear. Recently, a PCR assay based on the 16s ribosomal RNA gene sequence of M. pirum has been developed (30). This highly sensitive assay should help clarify studies of infection by this organism in patients with AIDS. Role for Mycoplasma in the Pathogenesis of Infections Mycoplasmas may be the only prokaryotic organisms that can grow “symbiotically” with eukaryotic cells without producing prominent cytotoxic effects or provoking extensive inflammatory responses in the host. Thus, clinically, many mycoplasmas can persist in a nonapparent state. Of interest, many disease processes associated with mycoplasmal infections are thought to be immunologically mediated (3 1,32). During the natural course of unrecognized mycoplasmal infections in humans, repeated episodes of infection superimposed on a nonapparent chronic infection may promote detrimental pathologic changes mediated by the immune system. In addition, persistent in-
8 1995 Elsevier Science Inc.
fections by these organism of seemingly low pathogenicity, may gradually but significantly alter the biology and physiology of infected hosts. Thus, these unique features of mycoplasmal infections may play an important role in1 many poorly understood chronic debilitating illnesses such as rheumatoid arthritis or AIDS. Development of significant clinical symptoms would require a long incubation time, thus masking the potential role of mycoplasmas in human diseases and making epidemiological studies difficult. Mycoplasmal infections and colonizations are often found in apparently healthy individuals. The possible pathogenic role played by many of these unusual microorganisms, including those previously considered commensals, should be reevaluated. Diagnosis Infections
of Mycoplasmal
Methods for the detection and identification of mycoplasma are difficult and time-consuming. Microbiological diagnosis typically depends on isolation of the microorganism in culture. Current culture systems for isolating fastidious mycoplasmas have had little success and often require prolonged incubation time. The usual histopathological techniques do not stain the wall-free mycoplasmas in clinical specimens. PCR, a highly sensitive diagnostic technique, has been applied to many experimental studies of mycoplasmal infections (33,34). However, preparation of clinical samples and each step of the amplification procedure must be monitored meticulously to prevent possible contamination and false-positive results. These potential problems have kept PCR as an experimental procedure and have prevented most clinical microbiology laboratories from using PCR as a general tool for diagnosis of mycoplasma infections. A sensitive and specific serologic assay such as ELISA or immunoblotting for rapid detection of mycoplasmal infections, similar to the techniques used in clinics for many viral infections, is urgently needed. Only when reliable mycoplasmal serological assays become available, will the true association between infections and dis-
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ease produced by these agents be recognized. The antigenic complexity of mycoplasmas is much greater than that of viruses. As such, the main challenge-in developing a useful serological assay is the ability to identify antibodies produced by the human host against specific target antigens of mycoplasmas. Furthermore, to be useful for diagnostic assays, the target antigens need to be uniquely specific for each species of mycoplasma. Because many Mycoplasm species are common inhabitants and considered resident flora in the human oropharyngeal and urogenital tracts, antibodies to them are likely to cross-react in serologic tests. Mycoplasmal lipid-associated membrane proteins are exposed on the microbial cell surface and are highly antigenic; therefore, they are the most likely immunogenic targets for the host’s response to infection. Recently we tested serum samples from 1,337 patients and showed that antibodies to lipid-associated membrane protein antigens of individual Myco; plasma spp. are highly specific and do not cross-react with those of other species (16,29). Thus, these protein antigens, each from different species of mycoplasmas, can be used to develop a sensitive and specific serologic assay. Treatment Infections
of Mycoplasmal
Diseases associated with mycoplasma may be amenable to proper antibiotic therapy. Although mycoplasmal infections can be suppressed by antibiotic therapy, they are difficult to eradicate. Treatment with erythromycin has significantly improved many clinical symptoms in patients with pneumonia caused by M. pneumoniae. However, the organisms may continue to be isolated from the respiratory tract of patients even after completion of antibiotic therapy. Successful eradication of the organisms relies on the hosts’ immune system in patients with normal immune function and treatment is complicated in patients who lack a functional immune system. Antibiotic susceptibility tests have shown that all clinical isolates of A4.fermentans are susceptible to ciproflox-
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acin, L-ofloxacin, and doxycycline but not erythromycin (35). Another study showed that M. penetruns was more susceptible to macrolide antibiotics (36). However, mycoplasmal infections in patients with AIDS are generally very refractory to antibiotic therapy. Rapid relapses of infection are experienced by immunocompromised patients once therapy is completed, even with high doses of antibiotics and a prolonged course of therapy. Selection of resistant strains following prolonged or multiple courses of antibiotic therapy, without eradication of the microorganisms, is a major concern. Conclusion Mycoplasmas are responsible for a variety of human infectious diseases. They cause many well-documented respiratory, genitourinary, and systemic illnesses. The actual role, if any, of mycoplasmas in AIDS remains unclear. We have presented three possibilities for their potential significance in AIDS patients: (i) mycoplasmas simply represent opportunistic infections found with high frequency in immunocompromised patients with AIDS; (ii) infections with mycoplasmas, such as M. fermentans and M. penetrans, may markedly enhance pathogenicity of human viruses including HIV-l, a phenomenon clearly demonstrated in in vitro studies; therefore, infections with some species of mycoplasma may promote disease progression to clinical AIDS; and (iii) the microbe itself is pathogenic in humans. This final possibility is supported by (i) our findings in an animal model of nonhuman primates, (ii) the association between M. fermentans infection and previously healthy non-AIDS patients dying of an acute disease, and (iii) association between M. penetrans infection and the development of Kaposi’s sarcoma in male homosexuals with AIDS. It is important to note that mycoplasmal infection is a highly significant cause of disease in AIDS patients, even if it is merely one more example of an opportunistic infection. Opportunistic infections are the direct cause of death in more than 80% of patients with AIDS. It is pathological to have an infectious (foreign) agent growing in the
Q 1995 Elsevier Science Inc.
liver, kidney, spleen, lung, brain, or other organ system. The detection of mycoplasmal infection becomes more significant if the microbe is acting as a disease-promoting “cofactor” ~_~or is itself responsible for certain aspects of AIDS pathogenesis. In view of the complexity of AIDS, we believe that the mycoplasmas may have a role in the above processes, but in any case, these unusual microorganisms deserve further study to better understand the pathogenesis of AIDS. Measurement of acute cytotoxic effects or acute patbogenicity.of the mycoplasmas that are of normally low virulence may not accurately, or at least not completely, indicate their possible roles in the pathogenesis of disease. To understand the possible significance of infection by some species of mycoplasma will depend on a better understanding of these extraordinary organisms including in so-called commensal states; Newly developed serological and molecular techniques should better equip scientists for the study of infections by these elusive microbes. References
Clyde, W.A., Jr. 1983.Mycoplasma pneumoniae respiratory disease symposium: summation and significance. Yale J. Biol. Med. 56523-527. Lo, S-C., et al. 1989. Association of the virus-like infectious agent originally reported in patients with AIDS with acute fatal disease in previously healthy nonAIDS patients. Am. J. Trop. Med. Hyg. 41:3&l-376. Simecka, J.W., et al. 1992. Mycoplasma diseases of animals, pp. 391416. In: J. Maniloff et al. (ed.), Mycoplasmas: molecular biology andpathogenesis. American Society for Microbiology, Washington, D.C. Lee, I.-M. and R.R. Davis. 1992. Mycoplasmas which infect plants and animals, pp. 379-390. In: J. Maniloff et al. (ed.), Mycoplasmas: molecular biology and pathogenesis. American Society for Microbiology, Washington, D.C. Krause, D.C. and D. Taylor-Robinson. 1992. Mycoplasmas which infect humans, pp. 4174-U. In: J. Maniloff et al. (ed.), Mycoplasmas: molecular biology and pathogenesis. American Society for Microbiology, Washington, D.C.
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6.
7.
8.
9.
Baum, S.G. 1994. Mycoplasma pneumoniae and atypical pneumonia, pp. 17041713. In: G.L. Mandell, LE. Bennett, and R. Dolin (ed.), Principles and practice of infectious diseases. Fourth edition. Churchill Livingstone, New York.
16. Wang, R.Y.-H., et al. 1993. Mycoplasma penetrans infection in male homosexuals with AIDS: high seroprevalence and association with Kaposi’s sarcoma. Clin. Infect. Dis. 17:724-729.
M&n& P.-A., and L. Westrom. 1970. Tubal and cervical cultures in acute salpingitis with special reference to Mycoplasma hominis and T-strain mycoplasmas. Br. J. Vener. Dis. 46:179-186.
17. Lo, S-C., et al. 1989. Identification of Mycoplasma incognitus infection in patients with AIDS: An immunohistochemical in situ hybridization and ultrastructural study. Am. J. Trop. Med. Hyg. 41601-616.
Thomsen, A.C. 1978. Occurrence of mycoplasmas in urinary tracts of patients with acute pyelonephritis. J. Clin. Microbial. 884-88.
18. Lo, S-C. 1992. Mycoplasmas and AIDS, pp. 525-545. In: J. Ma&off et al. (cd.), Mycoplasmas: molecular biology and pathogenesis. American Society for Microbiology, Washington, D.C.
Taylor-Robinson, D., and P. E. Munday. 1988. Mycoplasmal infection of the female genital tract and its complications, pp. 228-247. In: M.J. Hare (ed.), Genital tract infection in women. Churchill Livingstone, Edinburgh.
19. Bauer, F.A., et al. 1991. Mycoplasma fermentans (incognitus strain) infection in the kidneys of patients with acquired immunodeficiency syndrome and associated nephropathy: a light microscopic, immunohistochemical and ultrastructural study. Human Pathol. 22: 63-69.
10. Cassel, G.H., et al. 1983. Isolation of Mycoplasma hominis and Ureaplasma urealyticum from amniotic fluid at 1620 we& of gestation: potential effect on outcome of pregnancy. Sex. Transm. Dis. lO(Suppl.):294-302.
20. Ainsworth, J.G., et al. 1994. Mycoplasma fermentans and HIV-associated nephropathy. J. Infect. 29:323-326.
11. Kass, E.H., J.-S. Lin, and W. M:McCormack. 1986. Low birth weight and maternal colonization with genital mycoplasmas. Pediatr. Infect. Dis. 5(Suppl):279-281.
21. Dawson, M.S., et al. 1993. Detection and isolation of Mycoplasma fermentans from urine of HIV positive patients with AIDS. Arch. Pathol. Lab. Med. 117:511-514.
12. Tully, J.G., et al. 1981. A newly discovered mycoplasma in the human urogenital tract. The Lancet i:1288-1291.
22. Hawkins, R.E., et al. 1992. Association of mycoplasma and human immunodeficiency virus infection: detection of amplified Mycoplasma fermentans DNA in blood. J. Infect. Dis. 165:581-585.
13. Baseman, J.B., et al. 1988. Isolation and characterization of Mycoplasma genitalium strains from the human respiratory tract. J. Clin. Microbial. 26:22662269. 14. Homer, P.J., et al. 1993. Association of Mycoplasma genitaliwn with acute nongonococcal urethritis. The Lancet 342:582-585. 15. Montagnier, L., et al. 1990. A possible role of mycoplasmas as co-factors in AIDS, pp. P-17. In: M. Girard and L. Valette (ed.), Retroviruses of human AIDS and related animal diseases. Proc. Collogue des Cent Gardes. Lyons, France: Foundation M. Nerieux.
Clinical Micmbiology Newsletter 17:22,1995
23. Lo, S-C., et al. 1991. Enhancement of HIV-l cytocidal effects in CD4+ lymphocytes by the AIDS-associated mycoplasma. Science 25 1: 1074-1076. 24. Lemaitxe, M., et al. 1990. Protective activity of tetracycline analogs against the cytopathic effect of the human immunodeticiency viruses in CEM cells. Res. Viol. 1415-16. 25. Lo, S-C., et al. 1989. Association of the virus-like infectious agent originally reported in patients with AIDS with acute fatal disease in previously healthy nonAIDS patients. Am. J. Trop. Med. Hyg.
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41:364-376. 26. Lo, S.-C. 1993. Fatal systemic infections of non-human primates by Mycoplasma fermentans (incognitus strain). Clin. Infect. Dis. 17(Suppl l):S28%288. 27. Lo, S-C., et al. 1992. MycojGma penetrans. sp. nov., from the urogenital tract of patients with AIDS. Int. J. Syst. Bacteriol. 42:357-364. 28. Lo, S.-C., et al. 1993. Adhesion onto and invasion ofmammalian cells by Mycoplasmapenetrans-a newly isolated mycoplasma from patients with AIDS. Mod. Pathol. 6276-280. 29. Wang, R.Y.-H., et al. 1992. High frequency of antibodies to Mycoplasma penetrans in HIV-infected patients. The Lancet 340:1312-1316. 30. Grau, O., et al. 1993. Development of a selective and sensitive polymerase chain reaction assay for the detection of Mycoplasma pirum. FEMS Microbial. Lett. 106:327-334. 31. Cassell, G.H., and B. C. Cole. 1981. Mycoplasma as agents of human disease. N: Engl. J. Med. 304:80-89. 32. Lid, K. 1983. Manifestations and complications of Mycoplasma pneumoniae disease: a review. Yale. I. Biol. Med. 56461-468. 33. Wang, R.Y.-H. and S.-C. 1993. PCR detection of Mycopladma fermentans infection in blood and urine, pp. 511-516. In: D.H. Persing et al. (ed.), Diagnostic molecular microbiology, principles and applications. American Society for Microbiology, Washington, D. C. 34. Jensen, J.S., et al. chain reaction for plasma genital& J. Clii. Microbial.
1991. Polymerase detection of Mycoin clinical samples. 29:46-50.
35. Hayes, M.M., et al. 1993. In vitro antibi-’ otic susceptibility testing of different strains of Mycoplasma fermentans isolated from a variety of sources. Antimicrab. Agents Chemother. 37:2500-2503. 36. Poulin, S.A., R. E. Perkins, and R. B. Kundsin. 1994. Antibiotic susceptibilities of AIDS-associated mycoplasmas. 5. Clin. Microbial. 32:1101-l 103.
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New Understandings Shyh-Ching Lo, M.D., Ph.D. Chief, Molecular Pathobiology n_-_-.-._._. “J _ZI._l__r.‘ _.._am4 -..1 l-arawuc n_.._^:.:_ “eparwnem 1nJ”“‘“u-y . Disease Pathology Armed Forces Institute of Pathology Washington, D.C. 203066000
Mycoplasmas, a heterogeneous group of the smallest organisms capabie of seif-replication, beiong to the class Mollicutes. More than 100 different species of mycoplasmas have been identified. Like other bacteria, each species has distinct biological and biochemical properties associated with its ability to produce disease. The genetic material has the characteristics typical #.Fp‘ ...-tn...,,5*~” .-._..#.-a W&G o&n 1J :o “#llJ ,-..xl.. “1 “fial~“IGJ. Tlr,3 111G~Wlr”rrrG about 10 to 15% of that of usual bacteria, and genes necessary for cell-wall synthesis are absent. The wall-free organisms are highly pleomorphic in shape and are nutritionally fastidious, requiring rich, complex media to support their growth in culture. Specifically, all mycoplasmas need cholesterol, which is normally added as an animal serum supplement to the culture medium. Some Mollicutes, the Ureaplasma spp., can use urea as an energy source. Mycoplasmas are medically important bacteria res_ponsiblefor a wide range of human diseases. The infections they produce are commonly misdiagnosed as viral infections (1,2) because of the organisms’ small cell size, tilterable property, and the absence of acute inflammation that is atypical in bacterial infections. Mycoplasmas
in Animal Diseases
Mycoplasmal and ureaplasmal infecCzMNEEJ 17(22)x9-176.1995
November 15,1995
of Mycoplasmal
Infections and Disease
tions in animals are of historic importance in the field of mycoplasmology. The organisms have iong been recognized as pathogens causing significant health problems in commercial and laboratory animals. About a century ago, Mycoplasma mycoides was first described as the infectious agent causing ~~~r.Y~:,..~L-..:.., ,,..,..-,..:a 1‘a cvlllitgluus uu”lllG,1^.__ yKXl”yrrcur*r”‘ (CBPP) in cattle. Subsequently, mycoplasmas and ureaplasmas have been isolated from essentially every domestic and laboratory animal. Infections are often associated with the development of disease of the respiratory and urogenital tracts, joints, and other tissues I?). Mvconlasmaa significant im\_,_ -.-, __~__‘___ have ..-. _ _a _.~ .._ ___.._ -... pact on agricultural economy and biomedical research. In mycoplasmology, great emphasis has been and continues to be placed on the patbogenesis, diagnosis, and treatment of mycoplasma1 infections and diseases in animals. These wall-less prokaryotes also infect and cause disease in plants and insects (4). Ifis not clear whether the microorganisms can be transmitted to animals by infected insects with subsequent disease production.
Although mycoplasmas cause a variety of acute, fulminant, and chronic debilitating disease in animals, they are responsible for only a few significant human illnesses such as respiratory and urogenital tract infections (5). In fact, the most common community-acquired pneumonia is caused by Mycoplasma pneumoniae. The previously documented human mycoplasElsevier
ma1 diseases are briefly summarized here. M. pneutnoniae infection
This organism causes pneumonia in approximately 2 million people each year (6). The most prominent histopathological change associated with infection is interstitial pneumonitis with. edema and significant thickening of the septa. Unlike most bacterial pneumonias, leukocytosis and increased immature neutrophil (“band”) counts are generally not prominent in the atypical form of pneumonia. The main clinical symptoms at onset of M. pneumoniae infections are fever, malaise, and head-
In This Issue New Understandings of Mycoplasmal Infections and Disease.. . . . . . . . . . . . . . . . . . . . . .169 New diagnostic methods have given us a greater understanding of the scope of mycoplasma infections in healthy and immunocompromisedpeople
Regulatory Issues Affecting the Diagnostic Laboratory.. . . . . :. . ,174 The impact of increased federal regulation on the clinical laboratory and its personnel
Mycobacterium genavetzse and Mycobacteriuttt aviutn Mixed Infection in an AIDS Patient.. . . . . . 175 A case report 01%-4399/95/$0.00+ 09.sd
ache. The disease syndromes include pneumonia, tracheobronchitis, and pharyngitis. Many extrapulmonary infections, including synovitis, myocarditis, pericarditis, and a variety of central nervous system syndromes are also commonly described (6). It is not completely clear whether these complications are due to direct involvement of mycoplasmas in the diseased organ systems or are produced by immunologically mediated reactions. Mycoplusma hominis and Ureaplasma ureulyticum infections
Both-organisms colonize the lower urogenital tract of many healthy individuals; however, they have been implicated as the cause of pelvic inflammatory disease (7,8). M. hominis can be isolated in culture from 2 to 10% of healthy persons and from more than 60% of women with abnormal vaginal discharge. Septicemia due to M. hominis is responsible for postpartum fever or postabortion fever in women (9). M. hominis infections at many~extragenital sites have been reported including joints, respiratory tract, and central nervous system. Low birth weight (less than 1,000 g) infants infected with U. urealyticum have a significantly higher frequency of developing respiratory distress syndrome (lo), but it is less clear if women colonized with U. urealyticum in their urogenital tracts are at higher risk of giving birth to premature or low birthweight babies (11). Mycoplasma genitalium
infection
This highly fastidious mycoplasma has been recovered from the urogenital (12) and respiratory tracts (13) and has been implicated as a potential cause of pneumonia and nongonococcal urethritis (NGU). Because this mycoplasma is essentially uncultivable, it has been difficult to assess the scope of infection it
produces without a good seroepidemiological assay. PCR is currently the only diagnostic technique available. In a recent study by PCR, M. genital& was associated with NGU (14). Interest in M. genitalium increased after Montagnier and his associates (15) detected the organism by 'PCRassay in blood samples of patients with AIDS. A serological assay recently developed by us (see below) also detected a high frequency of M. genitalium infection in intravenous drug users and male homosexuals with or without HIV infection (16). However, few patients with hemophilia and AIDS had serologic evidence of M. genitalium infection. Most importantly, M. genital& was a rather common infection (>40%) among nonHIV-infected patients attending clinics for treatment of sexually transmitted diseases. Our study showed that the organism is the cause of a current epidemic of sexually transmitted disease that has not been previously detected. The organism is a likely candidate to produce systemic infections in the immunocompromised AIDS population.
Mycoplasmas in AIDS Recent studies in our laboratories and others have revealed that patients at high risk for developing AIDS have unusually high frequencies of infections by certain Mycoplasma spp. such as M. fermentans and M. penetrans (16-19). Infections with these mycoplasmas might play an important role in certain disease processes in AIDS patients (19, 20). M. fermentans, M. penetrans, and M. pirum have been identified much more frequently in patients infected with HIV-l, with or without AIDS, than in non-HIV-infected control subjects. Mycoplasma fermentans Our laboratory (21) and later Hawkins
and colleagues (22) reported that this organism is found most ~~_mm~nlv -, in --- the ---urogenital tracts or urine of patients at high risk for AIDS. Systemic infection with M. fermentans in patients__yith AIDS is also common and can be detected by PCR assays of urine, blood, tissue, and other clinical specimens. Importantly, infection appears to be associated directly with functional deficits of the infected organs’or tissues. For example, M. fermentans infection of the kidney is associated with the development of nephropathy in AIDS patients (19). Recently, an HIV-seropositive patient, who was not immunosuppressed, developed focal glomerulosclerosis, and M. fermentans DNA was found in the kidney biopsy by using PCR. M. fermentans was detected 15 mo later in peripheral blood lymphocytes, urine, and throat specimens. By 3 mo later, the CD4+ lymphocyte count had fallen and the patient developed Pneumocystis carinii pneumonia (20). This finding strengthened the association between M. fermentans and the development of AIDS nephropathy and has illustrated that _M.J’ fprmontnnr .v.ay . .... ..._..” infwtigfi ..._.,“l. precede rapid progression to AIDS. Mycoplasmas can markedly enhance cytocidal effects of HIV-l on human CD4+ lymphocytes in vitro (23.24). The organism is also associated with an acute form of adult respiratory distress syndrome that may produce systemic manifestations in persons who apparently were previously healthy (25,26). These observations suggest that M. fermentans may cause a heretofore unrecognized systemic form of human . mycoplasmal disease that might lead to a fatal outcome in nonimmunocompromised patients. Mycoplusma penetrans
In a study in our laboratory of urine samples obtained from 113 HIV-infected patients, this previously un--
NOTE: No responsibility is assumed by the Publisher for any injury and/or damage to persons or prop&y as a matter of products liability, negligence or otherwise, or from any use or ’ F nr ‘et “r pm&c&cc!4 be crti& au: *:n!e§s, i.? the ,z%ic:‘s pdgmciz, 5 iis:: i op.mtmo of zmy motto& p_md&, ms!r!zc!!oL _. i,ira. .I_ c-nn+s;n.=A __.._-. “I in .. . thr _.” mdr.4.l . ..” ._..... hrrrin . .. . . .. . . Nn .._ qpg+_l ” justiticd. Because of rapid advancea in medical sciences, we ncommcnd that the independent verification of diagnoses and drug dosages should be made. Discussions, views, and recommendations IWto medical procedures, choice of drugs, and dmg dosages are the responsibility of the authors. ClinicalMicrobiology Npwslener (ISSN 1069-417X) is issued twice monthly in one indexed volume per year by Elsevier Science Inc., 655 Avenue of the Americas, New York, NY 10010. Subscription price per year: $175.00 including postage and handling in the United States, Canada, and Mexico. Add $59.00 for postage in the rest of the world. Second-class postage paid at New York, NY and at additional mailing offkea. Postmastec Send address changes to Clinkol Microbiology Newsletter, Elsevier Science Inc., 655 Avenue of tbe Americas, New York, NY 1cKllO.
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Clinical Micmbiology Newsletter 17:22,1995
known human mycoplasma (27) was isolated more than 12 times from 6 patients. In a parallel study, it was not isolated from 98 HIV-negative, age-matched, healthy control individuals. The organism possesses a unique terminal structure by which it attaches to and penetrates eukaryotic cells. Extensive invasion of infected cells by M. penetrans produces cytopathic effects and may lead to cell death. By electron microscopic studies of urine sediments that grew M. penetrans in high titer, we determined that the organism, with its specialized tip structure, apparently adheres to the surface of and invades the cytoplasm of uroepithelial cells in the patient’s urogenital tract (28). Preliminary serologic studies using EIA and Western blot (immunoblot) analyses revealed a high prevalence (40%) of antibodies to M. penetrans in AIDS patients (29). In comparison, a low incidence (0.3%) of antibody is found in patients attending sexually transmitted disease clinics. In addition, of more than 150 HIV-negative patients with different non-AIDS-related disease states, including immune dysfunction and/or low leukocyte counts, none tested positive for M. penetrans antihodies. This newly discovered mycoplasma apparently is not a commensal organism in humans or just a simple opportunist commonly infecting immunocompromised hosts. It is uniquely associated with HIV- 1 infection and/or groups at high risk for AIDS (29). Subsequent serologic assays have revealed that the organism infects primarily male homosexuals with or without HIV infection (16). Few HIV-infected patients in other groups at high risk of AIDS are infected by this organism. Since Kaposi’s sarcoma occurs mainly in male homosexuals and not in other AIDS patients, a significant association can be made between serologic evidence of M. penefruns infection and the development of Kaposi’s sarcoma (p
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from rectal swabs of patients with AIDS, including those patients known to be urine culture-positive or seropositive for M. penetruns, were not successful. Mycopla.sma pirutn
This organism also has an organized terminal structure and metabolizes both glucose and arginine for growth. All isolates of M. pirum have been recovered from human cell cultures. The true origin or natural host for this species has not been well-documented. Reports from Montagnier and his associates (15) of the isolation of M. pirum from primary cultures of peripheral blood mononuclear cells from AIDS patients suggested that the organism is of human origin. The mycoplasma apparently has been isolated on two occasions from the blood of patients with AIDS. However, by using ELISA to examine for specific antibodies to M. pirum lipid-associated membrane proteins, we found few patients with AIDS or healthy non-HIV-infected individuals with serologic evidence of M. pirum infection (29). The significance of M. pirum in AIDS is still unclear. Recently, a PCR assay based on the 16s ribosomal RNA gene sequence of M. pirum has been developed (30). This highly sensitive assay should help clarify studies of infection by this organism in patients with AIDS. Role for Mycoplasma in the Pathogenesis of Infections Mycoplasmas may be the only prokaryotic organisms that can grow “symbiotically” with eukaryotic cells without producing prominent cytotoxic effects or provoking extensive inflammatory responses in the host. Thus, clinically, many mycoplasmas can persist in a nonapparent state. Of interest, many disease processes associated with mycoplasmal infections are thought to be immunologically mediated (3 1,32). During the natural course of unrecognized mycoplasmal infections in humans, repeated episodes of infection superimposed on a nonapparent chronic infection may promote detrimental pathologic changes mediated by the immune system. In addition, persistent in-
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fections by these organism of seemingly low pathogenicity, may gradually but significantly alter the biology and physiology of infected hosts. Thus, these unique features of mycoplasmal infections may play an important role in1 many poorly understood chronic debilitating illnesses such as rheumatoid arthritis or AIDS. Development of significant clinical symptoms would require a long incubation time, thus masking the potential role of mycoplasmas in human diseases and making epidemiological studies difficult. Mycoplasmal infections and colonizations are often found in apparently healthy individuals. The possible pathogenic role played by many of these unusual microorganisms, including those previously considered commensals, should be reevaluated. Diagnosis Infections
of Mycoplasmal
Methods for the detection and identification of mycoplasma are difficult and time-consuming. Microbiological diagnosis typically depends on isolation of the microorganism in culture. Current culture systems for isolating fastidious mycoplasmas have had little success and often require prolonged incubation time. The usual histopathological techniques do not stain the wall-free mycoplasmas in clinical specimens. PCR, a highly sensitive diagnostic technique, has been applied to many experimental studies of mycoplasmal infections (33,34). However, preparation of clinical samples and each step of the amplification procedure must be monitored meticulously to prevent possible contamination and false-positive results. These potential problems have kept PCR as an experimental procedure and have prevented most clinical microbiology laboratories from using PCR as a general tool for diagnosis of mycoplasma infections. A sensitive and specific serologic assay such as ELISA or immunoblotting for rapid detection of mycoplasmal infections, similar to the techniques used in clinics for many viral infections, is urgently needed. Only when reliable mycoplasmal serological assays become available, will the true association between infections and dis-
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ease produced by these agents be recognized. The antigenic complexity of mycoplasmas is much greater than that of viruses. As such, the main challenge-in developing a useful serological assay is the ability to identify antibodies produced by the human host against specific target antigens of mycoplasmas. Furthermore, to be useful for diagnostic assays, the target antigens need to be uniquely specific for each species of mycoplasma. Because many Mycoplasm species are common inhabitants and considered resident flora in the human oropharyngeal and urogenital tracts, antibodies to them are likely to cross-react in serologic tests. Mycoplasmal lipid-associated membrane proteins are exposed on the microbial cell surface and are highly antigenic; therefore, they are the most likely immunogenic targets for the host’s response to infection. Recently we tested serum samples from 1,337 patients and showed that antibodies to lipid-associated membrane protein antigens of individual Myco; plasma spp. are highly specific and do not cross-react with those of other species (16,29). Thus, these protein antigens, each from different species of mycoplasmas, can be used to develop a sensitive and specific serologic assay. Treatment Infections
of Mycoplasmal
Diseases associated with mycoplasma may be amenable to proper antibiotic therapy. Although mycoplasmal infections can be suppressed by antibiotic therapy, they are difficult to eradicate. Treatment with erythromycin has significantly improved many clinical symptoms in patients with pneumonia caused by M. pneumoniae. However, the organisms may continue to be isolated from the respiratory tract of patients even after completion of antibiotic therapy. Successful eradication of the organisms relies on the hosts’ immune system in patients with normal immune function and treatment is complicated in patients who lack a functional immune system. Antibiotic susceptibility tests have shown that all clinical isolates of A4.fermentans are susceptible to ciproflox-
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acin, L-ofloxacin, and doxycycline but not erythromycin (35). Another study showed that M. penetruns was more susceptible to macrolide antibiotics (36). However, mycoplasmal infections in patients with AIDS are generally very refractory to antibiotic therapy. Rapid relapses of infection are experienced by immunocompromised patients once therapy is completed, even with high doses of antibiotics and a prolonged course of therapy. Selection of resistant strains following prolonged or multiple courses of antibiotic therapy, without eradication of the microorganisms, is a major concern. Conclusion Mycoplasmas are responsible for a variety of human infectious diseases. They cause many well-documented respiratory, genitourinary, and systemic illnesses. The actual role, if any, of mycoplasmas in AIDS remains unclear. We have presented three possibilities for their potential significance in AIDS patients: (i) mycoplasmas simply represent opportunistic infections found with high frequency in immunocompromised patients with AIDS; (ii) infections with mycoplasmas, such as M. fermentans and M. penetrans, may markedly enhance pathogenicity of human viruses including HIV-l, a phenomenon clearly demonstrated in in vitro studies; therefore, infections with some species of mycoplasma may promote disease progression to clinical AIDS; and (iii) the microbe itself is pathogenic in humans. This final possibility is supported by (i) our findings in an animal model of nonhuman primates, (ii) the association between M. fermentans infection and previously healthy non-AIDS patients dying of an acute disease, and (iii) association between M. penetrans infection and the development of Kaposi’s sarcoma in male homosexuals with AIDS. It is important to note that mycoplasmal infection is a highly significant cause of disease in AIDS patients, even if it is merely one more example of an opportunistic infection. Opportunistic infections are the direct cause of death in more than 80% of patients with AIDS. It is pathological to have an infectious (foreign) agent growing in the
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liver, kidney, spleen, lung, brain, or other organ system. The detection of mycoplasmal infection becomes more significant if the microbe is acting as a disease-promoting “cofactor” ~_~or is itself responsible for certain aspects of AIDS pathogenesis. In view of the complexity of AIDS, we believe that the mycoplasmas may have a role in the above processes, but in any case, these unusual microorganisms deserve further study to better understand the pathogenesis of AIDS. Measurement of acute cytotoxic effects or acute patbogenicity.of the mycoplasmas that are of normally low virulence may not accurately, or at least not completely, indicate their possible roles in the pathogenesis of disease. To understand the possible significance of infection by some species of mycoplasma will depend on a better understanding of these extraordinary organisms including in so-called commensal states; Newly developed serological and molecular techniques should better equip scientists for the study of infections by these elusive microbes. References
Clyde, W.A., Jr. 1983.Mycoplasma pneumoniae respiratory disease symposium: summation and significance. Yale J. Biol. Med. 56523-527. Lo, S-C., et al. 1989. Association of the virus-like infectious agent originally reported in patients with AIDS with acute fatal disease in previously healthy nonAIDS patients. Am. J. Trop. Med. Hyg. 41:3&l-376. Simecka, J.W., et al. 1992. Mycoplasma diseases of animals, pp. 391416. In: J. Maniloff et al. (ed.), Mycoplasmas: molecular biology andpathogenesis. American Society for Microbiology, Washington, D.C. Lee, I.-M. and R.R. Davis. 1992. Mycoplasmas which infect plants and animals, pp. 379-390. In: J. Maniloff et al. (ed.), Mycoplasmas: molecular biology and pathogenesis. American Society for Microbiology, Washington, D.C. Krause, D.C. and D. Taylor-Robinson. 1992. Mycoplasmas which infect humans, pp. 4174-U. In: J. Maniloff et al. (ed.), Mycoplasmas: molecular biology and pathogenesis. American Society for Microbiology, Washington, D.C.
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Baum, S.G. 1994. Mycoplasma pneumoniae and atypical pneumonia, pp. 17041713. In: G.L. Mandell, LE. Bennett, and R. Dolin (ed.), Principles and practice of infectious diseases. Fourth edition. Churchill Livingstone, New York.
16. Wang, R.Y.-H., et al. 1993. Mycoplasma penetrans infection in male homosexuals with AIDS: high seroprevalence and association with Kaposi’s sarcoma. Clin. Infect. Dis. 17:724-729.
M&n& P.-A., and L. Westrom. 1970. Tubal and cervical cultures in acute salpingitis with special reference to Mycoplasma hominis and T-strain mycoplasmas. Br. J. Vener. Dis. 46:179-186.
17. Lo, S-C., et al. 1989. Identification of Mycoplasma incognitus infection in patients with AIDS: An immunohistochemical in situ hybridization and ultrastructural study. Am. J. Trop. Med. Hyg. 41601-616.
Thomsen, A.C. 1978. Occurrence of mycoplasmas in urinary tracts of patients with acute pyelonephritis. J. Clin. Microbial. 884-88.
18. Lo, S-C. 1992. Mycoplasmas and AIDS, pp. 525-545. In: J. Ma&off et al. (cd.), Mycoplasmas: molecular biology and pathogenesis. American Society for Microbiology, Washington, D.C.
Taylor-Robinson, D., and P. E. Munday. 1988. Mycoplasmal infection of the female genital tract and its complications, pp. 228-247. In: M.J. Hare (ed.), Genital tract infection in women. Churchill Livingstone, Edinburgh.
19. Bauer, F.A., et al. 1991. Mycoplasma fermentans (incognitus strain) infection in the kidneys of patients with acquired immunodeficiency syndrome and associated nephropathy: a light microscopic, immunohistochemical and ultrastructural study. Human Pathol. 22: 63-69.
10. Cassel, G.H., et al. 1983. Isolation of Mycoplasma hominis and Ureaplasma urealyticum from amniotic fluid at 1620 we& of gestation: potential effect on outcome of pregnancy. Sex. Transm. Dis. lO(Suppl.):294-302.
20. Ainsworth, J.G., et al. 1994. Mycoplasma fermentans and HIV-associated nephropathy. J. Infect. 29:323-326.
11. Kass, E.H., J.-S. Lin, and W. M:McCormack. 1986. Low birth weight and maternal colonization with genital mycoplasmas. Pediatr. Infect. Dis. 5(Suppl):279-281.
21. Dawson, M.S., et al. 1993. Detection and isolation of Mycoplasma fermentans from urine of HIV positive patients with AIDS. Arch. Pathol. Lab. Med. 117:511-514.
12. Tully, J.G., et al. 1981. A newly discovered mycoplasma in the human urogenital tract. The Lancet i:1288-1291.
22. Hawkins, R.E., et al. 1992. Association of mycoplasma and human immunodeficiency virus infection: detection of amplified Mycoplasma fermentans DNA in blood. J. Infect. Dis. 165:581-585.
13. Baseman, J.B., et al. 1988. Isolation and characterization of Mycoplasma genitalium strains from the human respiratory tract. J. Clin. Microbial. 26:22662269. 14. Homer, P.J., et al. 1993. Association of Mycoplasma genitaliwn with acute nongonococcal urethritis. The Lancet 342:582-585. 15. Montagnier, L., et al. 1990. A possible role of mycoplasmas as co-factors in AIDS, pp. P-17. In: M. Girard and L. Valette (ed.), Retroviruses of human AIDS and related animal diseases. Proc. Collogue des Cent Gardes. Lyons, France: Foundation M. Nerieux.
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23. Lo, S-C., et al. 1991. Enhancement of HIV-l cytocidal effects in CD4+ lymphocytes by the AIDS-associated mycoplasma. Science 25 1: 1074-1076. 24. Lemaitxe, M., et al. 1990. Protective activity of tetracycline analogs against the cytopathic effect of the human immunodeticiency viruses in CEM cells. Res. Viol. 1415-16. 25. Lo, S-C., et al. 1989. Association of the virus-like infectious agent originally reported in patients with AIDS with acute fatal disease in previously healthy nonAIDS patients. Am. J. Trop. Med. Hyg.
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41:364-376. 26. Lo, S.-C. 1993. Fatal systemic infections of non-human primates by Mycoplasma fermentans (incognitus strain). Clin. Infect. Dis. 17(Suppl l):S28%288. 27. Lo, S-C., et al. 1992. MycojGma penetrans. sp. nov., from the urogenital tract of patients with AIDS. Int. J. Syst. Bacteriol. 42:357-364. 28. Lo, S.-C., et al. 1993. Adhesion onto and invasion ofmammalian cells by Mycoplasmapenetrans-a newly isolated mycoplasma from patients with AIDS. Mod. Pathol. 6276-280. 29. Wang, R.Y.-H., et al. 1992. High frequency of antibodies to Mycoplasma penetrans in HIV-infected patients. The Lancet 340:1312-1316. 30. Grau, O., et al. 1993. Development of a selective and sensitive polymerase chain reaction assay for the detection of Mycoplasma pirum. FEMS Microbial. Lett. 106:327-334. 31. Cassell, G.H., and B. C. Cole. 1981. Mycoplasma as agents of human disease. N: Engl. J. Med. 304:80-89. 32. Lid, K. 1983. Manifestations and complications of Mycoplasma pneumoniae disease: a review. Yale. I. Biol. Med. 56461-468. 33. Wang, R.Y.-H. and S.-C. 1993. PCR detection of Mycopladma fermentans infection in blood and urine, pp. 511-516. In: D.H. Persing et al. (ed.), Diagnostic molecular microbiology, principles and applications. American Society for Microbiology, Washington, D. C. 34. Jensen, J.S., et al. chain reaction for plasma genital& J. Clii. Microbial.
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35. Hayes, M.M., et al. 1993. In vitro antibi-’ otic susceptibility testing of different strains of Mycoplasma fermentans isolated from a variety of sources. Antimicrab. Agents Chemother. 37:2500-2503. 36. Poulin, S.A., R. E. Perkins, and R. B. Kundsin. 1994. Antibiotic susceptibilities of AIDS-associated mycoplasmas. 5. Clin. Microbial. 32:1101-l 103.
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