Bacterial characterization by flow cytometry.

Bacteria were analyzed in a dual-beam flow cytometer after double staining with the fluorescent dyes chromomycin A3 and Hoechst 33258, which bind preferentially to DNA that is rich in guanine-cytosine and adenine-thymine, respectively. The measurements were indicative of the cellular DNA content and base composition, cell concentration, and proliferative state of the population. The ratio of the chromomycin A3 signal to the Hoechst 33258 signal increased with the guanine-cytosine content of the cellular DNA for the six cultured species measured, following expectation. Bacteria in urine from patients with urinary tract infections were characterized without interference from host cell DNA, debris, or other particulates.

Toxoplasma gondii pneumonitis in patients infected with the human immunodeficiency virus.

Pulmonary toxoplasmosis is a rarely recognized opportunistic infection in immunocompromised patients. A few case reports have described pulmonary toxoplasmosis in human immunodeficiency virus-infected patients in association with Toxoplasma gondii central nervous system disease. We encountered six cases of pulmonary toxoplasmosis in human immunodeficiency virus-infected patients who presented with a protracted febrile illness, respiratory symptoms, and an abnormal chest roentgenogram in the absence of neurologic findings. No clinical or roentgenographic features distinguished T gondii pneumonitis from more common opportunistic pulmonary infections. As the acquired immunodeficiency syndrome epidemic progresses, the presenting illnesses have evolved. Toxoplasma gondii must be considered a potential cause of pulmonary disease during the evaluation of human immunodeficiency virus-infected patients with respiratory symptoms.

Antibody to the retrovirus associated with the acquired immunodeficiency syndrome (AIDS). Presence in presumably healthy San Franciscans who died unexpectedly.

We performed autopsies and serologic tests in 189 subjects (152 men and 37 women) between 20 and 50 years of age with no history of immunosuppression who died unexpectedly and whose bodies were referred to the San Francisco coroner's office. Forty-eight of the 88 single men for whom addresses were available lived in areas of the city with a high incidence of the acquired immunodeficiency syndrome (AIDS). In addition, 36 of the subjects (30 men) were intravenous drug abusers. Antibody to the retrovirus associated with AIDS was present in 23 (18%) of the 121 subjects whose sera were tested. However, neither pathologic nor laboratory manifestations of AIDS were present in any of the 189 subjects who underwent autopsy. These results suggest that antibody to the retrovirus is common but subclinical manifestations of AIDS are uncommon in San Francisco, a city where the incidence of clinical AIDS is high.

The usefulness of diagnostic bone marrow examination in patients with human immunodeficiency virus (HIV) infection.

To determine the utility of bone marrow examination for the diagnosis of opportunistic infections and lymphoma in patients with known or suspected human immunodeficiency virus (HIV) infection, we retrospectively reviewed the medical and laboratory records of all patients undergoing diagnostic bone marrow examinations at San Francisco General Hospital between January 1, 1988 and December 31, 1989. All marrow examinations of patients with known or suspected HIV infection in which specimens were examined histopathologically and/or microbiologically for opportunistic pathogens or lymphoma were analyzed. Bone marrow examination resulted in the diagnosis of mycobacterial infection in 16% of the patients studied. Blood culture was 77% sensitive and bone marrow culture was 86% sensitive for detecting disseminated mycobacterial infection. This difference was not statistically significant (p greater than 0.05). Disseminated fungal infections occurred in less than 5% of the patients studied, and most were rapidly and accurately detected by examination of stained bone marrow samples. No case of lymphoma was diagnosed by bone marrow examination. Bone marrow examination may be useful for diagnosing opportunistic infections in patients with HIV infection. Mycobacterial blood cultures have a sensitivity comparable to bone marrow cultures in detecting disseminated mycobacterial infections, are less invasive, and may be less costly. Marrow examination is not useful for diagnosing lymphoma but can determine the extent of lymphoma that has been diagnosed by other means.

Rectal leishmaniasis in a patient with acquired immunodeficiency syndrome.

A severe rectal lesion due to Leishmania infection is described in an American-born homosexual man with the acquired immunodeficiency syndrome. The infection, which may have been venereally transmitted, responded to treatment with amphotericin B. There was no evidence of visceral leishmaniasis. The contribution of the patient's immunodeficiency to the development of the atypical cutaneous leishmanial lesion is unclear. The case may foretell increasing problems with protozoan infections in AIDS as the epidemic spreads to areas with endemic protozoan diseases.

Sulbactam/ampicillin versus metronidazole/gentamicin in the treatment of severe pelvic infections.

The clinical efficacy and safety of sulbactam/ampicillin versus metronidazole/gentamicin were compared in 39 patients with severe pelvic infections. 30 patients had severe acute pelvic inflammatory disease with peritonitis, 3 tubo-ovarian abscesses, 4 endomyometritis, and 2 posthysterectomy pelvic cellulitis. Aerobic and anaerobic cultures from the sites of infection yielded 259 micro-organisms from 38 patients; an average of 6.8 bacteria per infection (3.9 anaerobes and 2.9 aerobes). The most frequent isolates were Bacteroides spp. (21), B. bivius (13), B. disiens (8), Fusobacterium spp. (9), Peptostreptococcus anaerobius (15), P. asaccharolyticus (8), anaerobic Gram-positive cocci (17), Gardnerella vaginalis (24), Neisseria gonorrhoeae (14), alpha-haemolytic streptococci (6) and Escherichia coli (3). Clinical cure was noted in 19 of 20 patients treated with sulbactam/ampicillin and 16 of 19 treated with metronidazole/gentamicin. The sulbactam/ampicillin failure was a patient with pelvic inflammatory disease with a positive Chlamydia trachomatis culture who required antichlamydial therapy. The metronidazole/gentamicin failures included a patient with a tubo-ovarian abscess requiring surgical drainage and 2 patients with pelvic inflammatory disease requiring antichlamydial treatment. No adverse haematological, renal, or hepatic effects were noted with either regimen.

Use of the transbronchial biopsy for diagnosis of opportunistic pulmonary infections in acquired immunodeficiency syndrome (AIDS).

The authors used a method that includes Giemsa-stained touch preparations of a lung biopsy and culture of the same biopsy for viruses and microorganisms. The yield of etiologic agent diagnoses by this cytologic method was compared with yield from a simultaneous biopsy processed by usual histologic methods and stained by hematoxylin and eosin, silver methenamine, and other stains as necessary. Fifty-nine transbronchial biopsies and eight open lung biopsies were processed on 38 male patients with the clinical and laboratory features of acquired immunodeficiency syndrome (AIDS) to determine the etiology of their pulmonary disease. Pneumocystis carinii pneumonia was diagnosed in 16 patients. When sufficient material was available, the Giemsa-stained touch preparation agreed with the histologic examination. The touch preparation specimens were cultured, and seven of the patients with Pneumocystis carinii also had cytomegalovirus (CMV). Six additional patients had pulmonary CMV infection without evidence of Pneumocystis carinii pneumonia. Mycobacterial and fungal infections also were identified in these six patients. Sixteen patients had no specific diagnosis. With adequate material, their method provides reliable results and diagnosis within two to three hours of Pneumocystis carinii. In addition, it conserves the amount of specimen required, since the same material can be used for culture.

Etiology of acute salpingitis: influence of episode number and duration of symptoms.

The role of Neisseria gonorrhoeae in the etiology and pathogenesis of acute salpingitis and its relationship to nongonococcal salpingitis were investigated. To accomplish this goal, isolated microorganisms obtained from the fallopian tubes and cul-de-sac via laparoscopy were evaluated in relation to the number of episodes of salpingitis, duration of symptoms, and phase of menstrual cycle at infection onset. The incidence of isolation of N gonorrhoeae was inversely proportional to the number of episodes of salpingitis. No isolation of the gonococcus occurred from patients with 3 or more previous episodes of salpingitis. N gonorrhoeae was the most frequent organism recovered within the initial 24 hours of symptoms. Beyond 48 hours, the most frequent isolates were anaerobic bacteria, especially anaerobic cocci. Anaerobic bacteria were also recovered from the fallopian tubes in patients having their initial episode of salpingitis and within 24 hours of onset of symptoms. All fallopian tube isolates of gonococci were recovered within 7 days of the onset of menses.

The effect of polymethylmethacrylate and antibiotic combinations on bacterial viability. An in vitro and preliminary in vivo study.

Polymethylmethacrylate itself has a slight, long-term bacteriostatic effect on the growth of staphylococci, but while polymerizing the heat of polymerization and possibly the elaboration of monomer can be bactericidal to many pathogenic organisms. Twelve antibiotics incorporated into polymethylmethacrylate cement retained their antibacterial activity for long periods in dry storage, but the antibiotics leached out of polymethylmethacrylate quite rapidly in vitro and in vivo. No antibiotic could be found after thirty-seven days in treated cement pellets placed in a rabbit thigh.

Clofazimine crystals in alveolar macrophages from a patient with the acquired immunodeficiency syndrome.

An induced sputum specimen from a 35-year-old patient with the acquired immunodeficiency syndrome (AIDS) contained numerous bright orange-red needle-shaped crystal inclusions in his alveolar macrophages. Careful questioning revealed that he recently had been treated for 7 months with clofazimine (200 mg/d) for persistent Mycobacterium avium complex bacteremia. The striking cytologic finding observed is diagnosed easily if the characteristic morphologic appearance of the crystals and their location within the cytoplasm of macrophages and cells of the reticuloendothelial system is appreciated. Although this is the first observation at San Francisco (Calif) General Hospital of clofazimine crystals in a respiratory specimen from a patient with AIDS, the potential of more widespread therapy with clofazimine in patients with AIDS who are infected with M avium complex makes it imperative that the microscopic appearance of these crystals be recognized.

The use of mucolysed induced sputum for the identification of pulmonary pathogens associated with human immunodeficiency virus infection.

We describe a system for diagnosis of pulmonary disease in the human immunodeficiency virus-infected patient using induced sputum and other diagnostic procedures. This system has been successfully used at San Francisco (Calif) General Hospital for more than 2 years. It utilizes outpatient facilities and reduces the need for bronchoscopy. Sputum induced by inhalation of 3% saline mist, mucolysed, concentrated by centrifugation, and stained by a rapid modified Giemsa stain was the first diagnostic specimen examined in 404 episodes of suspected human immunodeficiency virus-associated pulmonary disease in 358 patients. Pneumocystis carinii was found in 222 (55%) sputum specimens. In 118 episodes in which the sputum did not contain P carinii, bronchoscopy with transbronchial biopsy and/or bronchoalveolar lavage was performed and P carinii was found in 50 (42%). These 118 bronchoscopy results, as well as evaluation of the subsequent clinical course of those patients who accounted for 64 episodes of lung disease and who did not have bronchoscopy following examination of nondiagnostic induced sputum, indicated a range of sensitivity for detection of P carinii in induced sputum of 74% to 77% and a negative predictive value of 58% to 64%. Mycobacteria were recovered from 11 (6%) of the induced sputum and 6 (12%) of the bronchoscopic specimens containing P carinii. However, only oral or environmental fungi were recovered from P carinii-containing induced sputum or bronchoscopic specimens. For those patients in whom P carinii was not detected, only the bronchoscopic specimens were cultured for Mycobacteria and fungi. Potentially pathogenic Mycobacteria and fungi were recovered from 16 (23.5%) and 34 (50%), respectively, of these P carinii-negative specimens. Analysis of these results, obtained under routine practice conditions, indicates that bronchoscopy should be reserved for those patients whose induced sputum examinations do not show P carinii and that mycobacterial and fungal cultures be performed only on bronchoscopic specimens in which P carinii is not detected.

Lack of effect of prophylactic aerosolized pentamidine on the detection of Pneumocystis carinii in induced sputum or bronchoalveolar lavage specimens.

Two independent studies were undertaken to determine the effect of prophylactic treatment with aerosolized pentamidine on the laboratory diagnosis of Pneumocystis carinii pneumonia in individuals at risk for or with the acquired immunodeficiency syndrome. The first study was a retrospective analysis to determine the effect of prophylactic treatment with aerosolized pentamidine on the diagnostic yield and sensitivity of detection of P carinii in induced sputum specimens. The results of examinations of 110 induced sputum specimens from patients who had not received aerosolized pentamidine were compared with the findings in 57 specimens from patients who had. There was no statistically significant difference between the two groups for the diagnostic yield in induced sputum specimens (48% vs 47%) or in bronchoalveolar lavage fluid specimens subsequently obtained from patients with nondiagnostic induced sputum examinations (33% vs 37%). The sensitivity of induced sputum specimens for identifying P carinii was 76% to 78% for patients who had not received aerosolized pentamidine and 71% to 75% for patients who had received the drug. The second study was a prospective comparison of 118 bronchoalveolar lavage fluid specimens to determine the effect of prophylactic treatment with aerosolized pentamidine on the number of organisms present. One hundred eighteen bronchoalveolar lavage fluid specimens were quantitatively examined and scored according to the number of clumps of P carinii present. No statistically significant difference was seen in the number of clumps of P carinii found in specimens from patients who had received aerosolized pentamidine vs the number of clumps found in specimens from patients who had not. In conclusion, prophylactic treatment with aerosolized pentamidine had no effect on (1) the diagnostic yield and sensitivity of detection of P carinii in induced sputum specimens or (2) the number of organisms detected in bronchoalveolar lavage fluid specimens obtained from individuals at risk for or with the acquired immunodeficiency syndrome.

Infection of the health-care worker by HIV and other blood-borne viruses: risks, protection, and education.

Transmission of the human immunodeficiency virus (HIV) and other blood-borne viruses in hospitals is discussed, and the infection control system and worker protection and education plan at San Francisco General Hospital (SFGH) are described. The acquired immunodeficiency syndrome (AIDS) epidemic has led to increased concern about occupationally acquired infections in health-care workers. As the number of HIV-infected persons increases, so does the risk of infection. Occupationally acquired HIV infection of health-care workers occurs principally in nurses, phlebotomists, and laboratory technicians through accidental subcutaneous injection of contaminated blood; splashing of blood onto open skin lesions, the eyes, and mucous membranes represents another route of exposure. The risk of infection from a single needle-stick exposure to HIV-infected blood is about 0.4%. Other blood-borne viruses to which employees are vulnerable include hepatitis B virus and human T-cell lymphotropic viruses, which may cause leukemia and lymphoma. SFGH has a comprehensive infection control system. Specimen containers are enclosed in transparent secondary containers, the worker is encouraged to wear protective clothing when necessary, and specific needle-stick precautions are promoted. There is also a health-care worker protection and education plan. The employee health services department provides immunizations, keeps records on accidental exposures, and operates a hot line. The education committee disseminates educational materials and arranges lectures. Infection control and education provide simple but effective measures for protecting hospital employees against HIV and other occupationally acquired infections.

In vitro correlates of delayed hypersensitivity in man: ambiguity of polymorphonuclear neutrophils as indicator cells in leukocyte migration test.

Delayed cutaneous hypersensitivity (DCH) of 12 normal adult subjects to purified protein derivative (PPD) of Mycobacterium tuberculosis, streptococcal streptokinase-streptodornase (SK-SD), and Candida albicans Dermatophytin O (DO) was assayed in vivo by skin testing and compared with such in vitro correlates of cellular immunity as lymphocyte transformation (LT) and inhibition of leukocyte migration (ILM) from microcapillary tubes or in agarose gel. LT was shown to be the best in vitro correlate of specific lymphocyte sensitization with all antigens. In the ILM assays, PPD showed good correlation with in vivo DCH and in vitro LT; SK-SD showed partial correlation; DO showed no correlation, not being active in any of the ILM tests. Cell distribution and morphology of stained migration patterns, ILM tests performed on separated populations of lymphocytes and polymorphonuclear leukocytes (PMN), as well as the ability of test antigens to stimulate PMN cells to reduce nitroblue-tetrazolium dye, indicated that in ILM tests mononuclear cells were not inhibited in their migration, whereas migration of PMN cells appeared to depend on their direct reaction with the test antigens.

Organization of microbiology laboratory services for the diagnosis of pulmonary infections in patients with human immunodeficiency virus infection.

When faced with the ever-increasing demand for the rapid laboratory diagnosis of Pneumocystis carinii pneumonia (PCP), as well as a variety of fungal, parasitic, and bacterial opportunistic infections in Human Immunodeficiency Virus (HIV)-infected individuals, the Chest Service and the Clinical Microbiology Laboratory at San Francisco General Hospital developed a coordinated plan for the collection and rapid examination of specimens to allow presumptive diagnoses to be made as quickly as possible. Clinical studies have been performed to determine the efficacy of different specimens and diagnostic systems for the provision of clinically useful information. Our approach has been to provide medical care on an outpatient basis using less invasive diagnostic systems that do not require direct physician participation. Direct laboratory examination of a single specimen submitted to microbiology often yielded presumptive or final diagnoses that were directly communicated to the physician. The evolution of PCP diagnosis in AIDS patients is described. Our current procedure of examining mucolysed, concentrated induced sputum stained with a rapid Giemsatype stain, Diff-Quik (Baxter/Scientific Products, McGaw, IL), has a sensitivity for P carinii detection of 74% to 77%. We have found that the use of commercially available immunofluorescence staining diminishes screening time and increases the sensitivity of P carinii detection in mucolysed concentrated induced sputum. We also describe the appearance of bacterial, fungal, and parasitic agents seen on direct examination and the usefulness of antigen detection, serology, and various microbiologic culture systems for diagnosing infections caused by these organisms.

Antigenic relationships and rapid identification of Peptostreptococcus species.

Antisera against whole cells of each Peptostreptococcus species (P. anaerobius, P. micros, P. parvulus, and P. productus) were produced in rabbits. When these antisera were reacted against sonically disrupted cells and culture supernatant fluids in Ouchterlony tests, lines of identity were obtained among the antigens from all the species and uninoculated culture medium. When the antisera were subsequently absorbed with the dehydrated culture medium used to grow the peptostreptococci, all cross-reactions in heterologous antigen-antibody combinations were eliminated, leaving only species-specific precipitin arcs. These absorbed antisera, specific for each Peptostreptococcus species by Ouchterlony tests, were used for rapid identification studies. Staphylococcus aureus-bearing protein A was sensitized with each absorbed antiserum. These reagents produced specific coagglutination reactions with suspensions of each Peptostreptococcus reference strain and with 16 clinical isolates. No cross-reactions occurred with the Streptococcus intermedius, Peptococcus magnus, or Peptococcus asaccharolyticus strains tested.