Enhancement of HIV-1 cytocidal effects in CD4+ lymphocytes by the AIDS-associated mycoplasma.

Coinfection with Mycoplasma fermentans (incognitus strain) enhances the ability of human immunodeficiency virus type-1 (HIV-1) to induce cytopathic effects on human T lymphocytes in vitro. Syncytium formation of HIV-infected T cells was essentially eliminated in the presence of M. fermentans (incognitus strain), despite prominent cell death. However, replication and production of HIV-1 particles continued during the coinfection. Furthermore, the supernatant from cultures coinfected with HIV-1 and the mycoplasma contained a factor that inhibited the standard reverse transcriptase enzyme assay. The modification of the biological properties of HIV-1 by coinfection with mycoplasma may be involved in the pathogenesis of acquired immunodeficiency syndrome (AIDS).

Cat scratch disease: a bacterial infection.

Histopathologic examination of lymph nodes from 39 patients with clinical and pathological criteria for cat scratch disease revealed delicate pleomorphic Gram-negative bacilli in 34 of the 39 nodes. They were within the walls of capillaries in or near areas of follicular hyperplasia and within microabscesses. They were best seen with the Warthin-Starry silver impregnation stain. Organisms in lymph node sections exposed to convalescent serum from three patients and to immunoperoxidase stained equally well with all three samples. The organisms did not react with hyperimmune sera to Legionella pneumophila nor to several species of Rickettsia. These bacilli appear to be the causative agents of cat scratch disease.

Culture-proved disseminated cat-scratch disease in acquired immunodeficiency syndrome.

A male homosexual (positive for the human immunodeficiency virus) with a recent cat scratch developed fever, epitrochlear and axillary lymphadenopathy, and retinitis. Subsequently, he developed skin (epitheloid hemangioma) and mucosal lesions (Kaposi's sarcoma), multiple liver abscesses, and pleural effusion. Warthin-Starry stains and/or electron micrographs of lymph nodes and skin lesions demonstrated bacilli characteristic of those associated with cat-scratch disease. Cultures of lymph node, pleural fluid, and liver abscess specimens yielded organisms believed to be the causative agent of cat-scratch disease. We believe that disseminated cat-scratch disease may become an indicator of opportunistic infection signaling acquired immunodeficiency syndrome in a patient who is positive for the human immunodeficiency virus.

Life-threatening cat-scratch disease in an immunocompromised host.

We describe a renal allograft recipient with cat-scratch disease in whom refractory hypotension, severe metabolic acidosis, pulmonary infiltrates, and encephalopathy developed. The patient first presented with a history of cat bites and scratches, fever, headache, and arthralgias. Four weeks later, the clinical presentation of septic shock suddenly developed in the patient. Cat-scratch disease was documented clinically and by finding delicate pleomorphic bacilli in Warthin-Starry silver stains of biopsy specimens taken from the primary inoculation site and regional lymph node. The administration of intravenous sulfamethoxazole and trimethoprim, erythromycin lactobionate, and tobramycin sulfate therapy correlated with recovery. Although cat-scratch disease is usually a benign, self-limited illness, this article illustrates its systemic nature, its potential for devastating complications in the immunocompromised host, and its possible response to vigorous antibiotic therapy.

Mycoplasma fermentans (incognitus strain) infection in the kidneys of patients with acquired immunodeficiency syndrome and associated nephropathy: a light microscopic, immunohistochemical, and ultrastructural study.

We studied renal tissues from 203 patients with acquired immunodeficiency syndrome (AIDS). Of the 203 patients, 20 showed light-microscopic changes characteristic of AIDS-associated nephropathy (AAN). Fifteen of the 20 (group A) were examined by immunohistochemistry using Mycoplasma fermentans (incognitus strain)-specific monoclonal antibodies and electron microscopy. Renal tissues from all 15 AAN patients showed positive staining for the incognitus strain mycoplasmal antigens within glomerular endothelial and epithelial cells, glomerular basement membrane, tubular epithelial cells and casts, and mononuclear interstitial cells. Ultrastructural study of these 15 cases revealed mycoplasma-like structures in various locations including glomerular epithelial and endothelial cells, glomerular basement membrane, tubular epithelial cells and casts, and mononuclear interstitial cells. In a parallel study, renal tissues from 15 patients with AIDS with essentially normal renal histology or mild interstitial mononuclear cell infiltration (group B) were also examined. These tissues showed no evidence of incognitus strain mycoplasmal infection in renal parenchymal cells; however, occasional scattered mononuclear interstitial cells were positive for the antigens of this organism. Renal tissues from five patients dying with non-AIDS diseases (group C) showed no staining for the incognitus strain antigens in any location. Therefore, infection of renal parenchymal cells by M fermentans (incognitus strain) in the kidneys of AIDS patients is apparently associated with AAN.

Association of the virus-like infectious agent originally reported in patients with AIDS with acute fatal disease in previously healthy non-AIDS patients.

We studied 6 patients from 6 different geographic areas who presented with acute flu-like illnesses. The patients developed persistent fevers, lymphadenopathy or diarrhea, pneumonia, and/or heart, liver, or adrenal failure. They died in 1-7 weeks. These patients had no serological evidence of HIV infection and could not be classified as AIDS patients according to CDC criteria. The clinical signs as well as laboratory and pathological studies of these patients suggested an active infectious process, although no etiological agent was found despite extensive infectious disease work-ups during their hospitalization. Post-mortem examinations showed histopathological lesions of fulminant necrosis involving the lymph nodes, spleen, lungs, liver, adrenal glands, heart, and/or brain. No viral inclusion cells, bacteria, fungi, or parasites could be identified in these tissues using special tissue stains. We report that immunohistochemistry using rabbit antiserum raised against VLIA, the virus-like infectious agent previously identified in patients with AIDS and shown to cause fatal systemic infection in primates, revealed VLIA antigens in these necrotizing lesions. In situ hybridization using an 35S labeled VLIA-specific DNA probe also detected VLIA genetic material in the areas of necrosis. Furthermore, virus-like particles closely resembling VLIA were identified ultrastructurally in these histopathological lesions. VLIA was associated with the systemic necrotizing lesions in these previously healthy non-AIDS patients with an acute fatal disease.

Virus-like infectious agent (VLIA) is a novel pathogenic mycoplasma: Mycoplasma incognitus.

The newly recognized pathogenic virus-like infectious agent (VLIA), originally reported in patients with AIDS but also known to be pathogenic in previously healthy non-AIDS patients and in non-human primates, was cultured in cell-free conditions using a modified SP-4 medium and classified as a member of the order Mycoplasmatales, class Mollicutes. The infectious microorganism is tentatively referred to as Mycoplasma incognitus. M. incognitus has the unique biochemical properties of utilizing glucose both aerobically and anaerobically, as well as having the ability to metabolize arginine. Among all known human mycoplasmas, these specific biochemical characteristics were found previously only in a rarely isolated species, M. fermentans. In comparison with M. fermentans, M. incognitus appears to be even more fastidious in cultivation requirements and fails to grow in all tested mycoplasma media other than modified SP-4 medium. In addition, M. incognitus grows much more slowly, has a smaller spherical particle size and occasional filamentous morphology, and forms only irregular and very small colonies with diffuse edges on agar plates. Antigenic analysis using polyclonal and monoclonal antibodies and DNA analysis of sequence homology and restriction enzyme mappings in M. incognitus, M. orale, M. hyorhinis, M. hominis, M. pneumoniae, M. fermentans, M. arginini, M. genitalium, M. salivarium, Ureaplasma urealyticum, and Acholeplasma laidlawii revealed that M. incognitus is distinct from other mycoplasmas, but is most closely related to M. fermentans.

Identification of Mycoplasma incognitus infection in patients with AIDS: an immunohistochemical, in situ hybridization and ultrastructural study.

Monoclonal antibodies (Mabs) were developed against antigens from a pure culture of Mycoplasma incognitus grown in modified SP-4 medium. All the Mabs obtained were shown to react only with M. incognitus, and not with other species of human mycoplasma. The Mabs identified M. incognitus immunohistologically in thymus, liver, spleen, lymph node, or brain from 22 patients with AIDS, as well as in 2 placentas delivered by patients with AIDS. Using an 35S-labeled DNA probe specific for M. incognitus and in situ hybridization technique, we also identified M. incognitus-specific genetic material in these tissues. Furthermore, ultrastructural studies of the specific areas of tissues which were highly positive for M. incognitus antigens revealed characteristic structures of mycoplasma organisms. These mycoplasma-like particles could be identified intracellularly and extracellularly. Histopathology of the tissues infected by M. incognitus varied from no pathological changes to fulminant necrosis with or without an associated inflammatory reaction. M. incognitus, a novel pathogenic mycoplasma, was cytopathic and cytocidal.

Mycoplasmal infections alter gene expression in cultured human prostatic and cervical epithelial cells.

To better understand how infections by mycoplasmas affect gene expression in human cells, we quantitatively measured the transcripts of 38 cytokine genes in HPV E6- and E7-immortalized cervical and prostatic epithelial cells before and after infection by four human urogenital mycoplasmas, M. fermentans, M. genitalium, M. hominis and M. penetrans. Using the multi-probe RNase protection assay (RPA), 22 and 23 cytokine gene transcripts were detected in the non-infected control prostatic and cervical epithelial cells, respectively. Although there were no discernible changes in cell morphology and growth kinetics following 72 h of mycoplasmal infection, 55-74% of the cytokine genes expressed in the two human epithelial cell lines were altered. Most changes reflected an increased expression of these cytokine genes, while expression of some cytokine genes significantly decreased. The effects varied with host cell type and species of infecting mycoplasmas. These alterations in gene expression were more profound in the cervical epithelial cells than in the prostatic cells. M. fermentans produced the most significant effects, followed by M. penetrans, M. genitalium and M. hominis. Some alterations in the gene expression were transient, but most persisted over the course of chronic (9 months) mycoplasmal infection. Prolonged gene expression changes induced by chronic mycoplasmal infection may gradually alter important biological properties in the infected mammalian cells and produce a unique form of disease process.

Cat-scratch disease masquerading as a solitary tumor of the breast.

Cat-scratch disease in breast lymph node tissue may present as a mass lesion and require clinical investigation to rule out malignancy. Four cases involving prepectoral lymph nodes have been diagnosed at the Armed Forces Institute of Pathology, Washington, DC. Mammographic findings of a smooth-edged soft-tissue density without spiculation or calcification may suggest the diagnosis. Warthin-Starry silver impregnation of tissue sections is the preferred method of diagnosis, and should be added to the diagnostic workup of granulomatous lesions of the breast.

Acid-fastness of fungi in blastomycosis and histoplasmosis.

In 60% of the infections caused by Blastomyces dermatitidis and in 47% of the infections caused by Histoplasma capsulatum, some of the organisms are acid-fast in tissue sections. Treatment with 1N hydrochloric acid at 60 degrees C abolishes the acid-fastness of H capsulatum, which indicates that the acid-fastness may be related to mycolic acid. Organisms of B dermatitidis, however, remain acid-fast in 83% of the sections treated with acid. The presence of acid-fastness in small cells can aid in the differentiation of B dermatitidis and H capsulatum from Cryptococcus neoformans and Candida albicans, Persistent acid-fastness of organisms after treatment with heated acid helps identify B dermatitidis in tissue sections, especially when the strain is small or in other ways atypical.

In vitro antimicrobial susceptibility testing for the newly identified AIDS-associated Mycoplasma. Mycoplasma fermentans (incognitus strain).

Mycoplasma fermentans (incognitus strain) has recently been recognized as a possible infectious pathogen in humans. This mycoplasma is associated with an acute fatal disease in previously healthy patients who do not have the acquired immunodeficiency syndrome. Many patients with the acquired immunodeficiency syndrome suffer a systemic infection with this microbe. Quantitative assay of antimicrobial susceptibility for M fermentans (incognitus strain) in cultures to representative antibiotics has revealed that the microbe is not sensitive to erythromycin, the most commonly used antibiotic for human mycoplasma infections. The testing shows that M fermentans (incognitus strain) is sensitive in vitro to the antibiotics tetracycline, doxycycline, chloramphenicol, clindamycin, lincomycin, and ciprofloxacin.

Systemic cat scratch disease: report of 23 patients with prolonged or recurrent severe bacterial infection.

Over a seven-year period, we identified 23 patients who had prolonged or recurrent, severe, systemic, cat-scratch disease (CSD). Compared with the usual, benign course in 1,038 patients with typical CSD, the course in these 23 patients included prolonged (two or more weeks) morbidity (fever, malaise, fatigue, myalgia, arthralgia, skin eruptions, weight loss, and splenomegaly). Five patients with systemic CSD had either neuroretinitis, pleurisy, arthralgia or arthritis, splenic abscesses, and mediastinal masses or enlarged nodes of the head of the pancreas. Recurrent CSD in two of three adults was confirmed by finding typical CSD bacilli in lymph nodes removed during separate episodes. The majority of patients were adult males, and all patients recovered completely without sequelae. Histopathologic studies of five skin and 18 lymph node biopsy specimens were diagnostic. CSD bacilli were detected in lymph nodes from 15 patients and in the primary skin lesions of four patients. CSD bacilli were found in both skin and lymph nodes of three patients.

Intraoral Hodgkin's disease.

An 85-year-old woman presented with an intraoral lesion involving the edentulous soft tissue of the mandibular alveolar crest between the first premolar and first molar teeth. Diagnosis was made with the aid of a biopsy. The gallium scan and CT scan were used successfully for noninvasive staging. However, initial treatment was delayed due to the patient's wishes, overall poor health, and decreased tolerance to both radiation and chemotherapy. This resulted in spread of the disease leading to her death.

Demonstration of Chlamydia trachomatis in inguinal lymphadenitis of lymphogranuloma venereum: a light microscopy, electron microscopy and polymerase chain reaction study.

Intravacuolar organisms in vacuolated macrophages were associated with areas of necrosis and suppuration in 12 patients with suppurative inguinal lymphadenitis. The intravacuolar organisms measured 0.2 to 2.0 micrometers in diameter, stained Gram negative with the Brown-Hopp's tissue Gram stain, faintly blue with hematoxylin and eosin stain, and black with the Warthin-Starry silver impregnation stain. The organisms lined vacuolar membranes and/or clumped in centers of vacuoles. Electron microscopy revealed elementary and reticulate bodies and intermediate forms characteristic of the genus Chlamydia. Cultures of three lymph nodes in McCoy cells grew Chlamydia trachomatis, lymphogranuloma venereum (LGV) serovars. Polymerase chain reaction using primers for chlamydial 16S ribosomal DNA confirmed the organisms as Chlamydia in lymph nodes from nine patients. Recognition of chlamydial organisms by light microscopy in tissue sections of lymph nodes allows a definitive diagnosis of lymphogranuloma venereum.

In vitro antibiotic susceptibility testing of different strains of Mycoplasma fermentans isolated from a variety of sources.

The in vitro susceptibilities to antibiotics of 24 strains of Mycoplasma fermentans (isolated from human immunodeficiency virus type 1-infected AIDS patients, non-AIDS patients with acute respiratory disease, and tissue culture) were determined. MICs for 90% of the strains tested (micrograms per milliliter) were obtained for chloramphenicol (1.25), ciprofloxacin (0.078), clindamycin (0.078), doxycycline (0.625), erythromycin (> 10), gentamicin (> 10), levofloxacin (0.078), lincomycin (0.156), streptomycin (> 10), and tetracycline (0.625).

High-level expression of H-ras and c-myc oncogenes in mycoplasma-mediated malignant cell transformation.

C3H mouse embryo cells, which normally have low inherent spontaneous transformation, underwent malignant transformation while chronically infected with Mycoplasma fermentans or Mycoplasma penetrans. This mycoplasma-mediated oncogenic process had long latency (more than 7 weeks of persistent mycoplasmal infection) and showed multistage progression characterized by reversibility and irreversibility of malignant properties upon removal of M. fermentans from culture. Marked expression of H-ras and c-myc mRNA, but not N-myc, src, N-ras, or p53 mRNA, was found in the mycoplasma-transformed C3H cells that exhibited characteristic malignant properties of morphological changes and uncontrolled cell growth. However, at least up to the eleventh week of persistent mycoplasma infection, the marked expression of H-ras or c-myc mRNA in C3H cells depended on continued presence of the mycoplasma in culture. H-ras or c-myc mRNA rapidly declined to the undetectable low levels of nontransformed parental C3H cells, and all malignant properties of the once-fully-transformed C3H cells quickly reversed, if M. fermentans was eradicated from culture. In comparison, infection with M. penetrans for 7 or 11 weeks also induced a high level of H-ras, but not c-myc, mRNA expression in C3H cells. Despite having prominent amount of steady-state H-ras mRNA, these M. penetrans-infected C3H cells did not show any sign of malignant transformation. Thus, marked expression of H-ras gene alone was not sufficient to effect transformation in C3H cells. Interestingly, after a further prolonged (18 weeks) infection with either M. fermentans or M. penetrans, C3H cells revealed prominent chromosomal changes, expressed constitutively (with or without the presence of the transforming mycoplasmas) at high levels of both H-ras and c-myc mRNA and became permanently transformed. These cells were able to form tumors in animals.

Mycoplasmas and oncogenesis: persistent infection and multistage malignant transformation.

Oncogenic potential of human mycoplasmas was studied using cultured mouse embryo cells, C3H/10T1/2 (C3H). Mycoplasma fermentans and Mycoplasma penetrans, mycoplasmas found in unusually high frequencies among patients with AIDS, were examined. Instead of acute transformation, a multistage process in promotion and progression of malignant cell transformation with long latency was noted; after 6 passages (1 wk per passage) of persistent infection with M. fermentans, C3H cells exhibited phenotypic changes with malignant characteristics that became progressively more prominent with further prolonged infection. Up to at least the 11th passage, all malignant changes were reversible if mycoplasmas were eradicated by antibiotic treatment. Further persistent infection with the mycoplasmas until 18 passages resulted in an irreversible form of transformation that included the ability to form tumors in animals and high soft agar cloning efficiency. Whereas chromosomal loss and translocational changes in C3H cells infected by either mycoplasma during the reversible stage were not prominent, the onset of the irreversible phase of transformation coincided with such karyotypic alteration. Genetic instability--i.e., prominent chromosomal alteration of permanently transformed cells--was most likely caused by mutation of a gene(s) responsible for fidelity of DNA replication or repair. Once induced, chromosomal alterations continued to accumulate both in cultured cells and in animals without the continued presence of the transforming microbes. Mycoplasma-mediated multistage oncogenesis exhibited here shares many characteristics found in the development of human cancer.