Clinical correlation of variations in the internal transcribed spacer regions of rRNA genes in Pneumocystis carinii f.sp. hominis.

OBJECTIVES: To analyse the importance of sequence variations in the internal transcribed spacer (ITS) regions 1 and 2 of the nuclear rRNA operon in AIDS patients with Pneumocystis carinii pneumonia (PCP). DESIGN AND METHODS: ITS 1 and 2 genotypes were determined in 162 bronchoalveolar lavage samples from 130 patients participating in a prospective cohort study of PCP. RESULTS: A total of 49 different ITS genotypes were detected. ITS genotype was not associated with the clinical severity or outcome of PCP. In 37 of 162 (23%) samples infection with two or more genotypes was observed. A genotype switch was detected in six of 10 patients (60%) with recurrent episodes of PCP. However, genotype changes were also seen in 10 of 19 patients (53%) who had repeated bronchoscopies within the same episode of PCP. The same ITS type was observed twice in 13 (46%) of the 28 patients with repeat bronchoscopies during single or recurrent episodes of pneumonia, but in only 14 of 81 (17%) randomly selected pairs (P < 0.01). CONCLUSION: Although the detection of ITS genotypes is not a random event, changes in genotype can be detected in a single episode of disease, with 23% of PCP patients being infected with more than one P. carinii genotype, thus complicating the use of this locus as a genetic marker to separate new infection from the reactivation of latent infection. ITS genotypes are not associated with the clinical severity of PCP.

Heterozygosity for a deletion in the CKR-5 gene leads to prolonged AIDS-free survival and slower CD4 T-cell decline in a cohort of HIV-seropositive individuals.

OBJECTIVE: Recently, it has been shown that a homozygous 32 base-pair deletion in the gene encoding CKR-5, a major coreceptor for HIV-1, leads to resistance to infection with HIV-1. We have investigated whether HIV-seropositive individuals who were heterozygous for the CKR-5 deletion had a different course of the disease. DESIGN: Thirty-five high-risk HIV-seronegative and 99 HIV-seropositive Danish homosexual men followed form 1985 to 1996 and 37 blood donors were analysed for their CKR-5 genotype by polymerase chain reaction. RESULTS: Two (6%) of the 35 HIV-seropositive subjects at high-risk of infection were homozygous and seven (20%) were heterozygous for the CKR-5 deletion. This was not significantly different from the distribution in normal donors. Twenty-two (22%) of the 99 HIV-seropositive subjects were heterozygous and none was homozygous. Two subgroups of patients who had an opposite course of the HIV disease were identified. Of nine long-term non-progressors, six (66%) were heterozygous for the deletion. This frequency is significantly higher than in nine rapid progressors of whom non was heterozygous. The frequency of heterozygotes in long-term non-progressors was also significantly higher than in the cohort as a whole. A Kaplan-Meier plot of the HIV-seropositive subjects, of whom 57 developed AIDS, showed a significantly better prognosis within the first 7 years of follow-up for those who were heterozygous for the deletion. Heterozygous individuals also had a significantly slower decrease in CD4 T-cell count per year. CONCLUSION: Individuals who are heterozygous for the 32-base-pair deletion in the CKR-5 gene have a slower decrease in their CD4 T-cell count and a longer AIDS-free survival than individuals with the wild-type gene for up to 11 years of follow-up.

Prognostic markers of short-term mortality in AIDS-associated Pneumocystis carinii pneumonia.

BACKGROUND: Since 1990, corticosteroids have been recommended as adjunctive therapy for patients with AIDS-associated Pneumocystis carinii pneumonia (PCP) and respiratory failure. We hypothesized that the natural course of AIDS-associated PCP has changed in the era of adjunctive corticosteroid therapy. OBJECTIVE: To study variables obtained on hospital admission for possible prognostic value of short-term (3-month) outcome of PCP. DESIGN AND PATIENTS: Prospective observational study of 176 consecutive HIV-1-infected individuals with PCP between 1990 and 1999. METHOD: Cox proportional-hazards regression models. RESULTS: Univariate analysis showed that age, one or more prior episodes of PCP, use of antimicrobial therapy other than trimethoprim-sulfamethoxazole (TMP-SMZ), use of PCP prophylaxis at diagnosis, and culture of cytomegalovirus (CMV) in BAL predicted progression to death within 3 months. After adjustment, age (relative risk [RR], 4.1; 95% confidence interval [CI], 1.8 to 9.3), initial antimicrobial therapy other than TMP-SMZ (RR, 3.1; 95% CI, 1.2 to 8.5), use of PCP prophylaxis (RR, 5.6; 95% CI, 2.2 to 14.4), and culture of CMV in BAL fluid (RR, 2.7; 95% CI, 1.3 to 5.6) remained independent predictors of a poor outcome. In contrast, neither PO(2) nor serum lactate dehydrogenase, which in earlier studies were identified as prognostic markers, were predictors of mortality. CONCLUSION: Age, initial anti-PCP therapy, use of PCP prophylaxis, and BAL CMV status may be useful predictors of outcome of PCP in patients treated in the era of adjunctive corticosteroid therapy.

Alveolar damage in AIDS-related Pneumocystis carinii pneumonia.

OBJECTIVE: Pneumocystis carinii pneumonia is the most common and serious of the pulmonary complications of AIDS. Despite this, many basic aspects in the pathogenesis of HIV-associated P carinii pneumonia are unknown. We therefore undertook a light and electron microscopic study of transbronchial biopsy specimens to compare pathologic features of P carinii pneumonia and other HIV-related lung diseases. DESIGN AND PATIENTS: Thirty-seven consecutive HIV-infected patients undergoing a diagnostic bronchoscopy. RESULTS: P carinii pneumonia was characterized by an increase in inflammation, edema, exudate, fibrosts, type II pneumocyte proliferation, and cellular infiltration of the alveolar wall when compared with other lung diseases (all p < 0.05). Electron microscopy showed apposition of the trophozoite to the type I pneumocyte. Erosion of type I pneumocytes was observed in 13 of 15 patients with P carinii pneumonia, whereas none without P carinii pneumonia had this finding (p < 0.05). Erosion of the type II pneumocyte was not observed. CONCLUSION: Inflammation, interstitial fibrosis, and alveolar epithelial erosion are characteristic features of P carinii pneumonia. The changes may form the pathologic basis for the respiratory failure seen in patients with P carinii pneumonia. Electron microscopy did not show any diagnostie advantage over conventional light microscopy using routine stains.

Interleukin-8 and leukotriene B4 in bronchoalveolar lavage fluid from HIV-infected patients with bacterial pneumonia.

Human immunodeficiency virus (HIV)-infected patients are at increased risk of contracting bacterial infections, mainly pneumonia. Despite this, little is known about immunopathogenic mechanisms in HIV-related bacterial pneumonia. This paper investigates the presence of the neutrophil chemotactic mediators, interleukin-8 (IL_8) and leukotriene B4 (LTB4), in bronchoalveolar lavage (BAL) fluid from 27 HIV-infected patients with bacterial pneumonia. Significantly elevated levels of IL-8 were found in BAL fluid of patients with bacterial pneumonia [529 pg ml-1 (296-1161 pg ml-1)] compared to matched patients with Pneumocystis carinii pneumonia (PCP) [59 pg ml-1 (42-254 pg ml-1)] and healthy controls [58 pg ml-1 (37-82 pg ml-1)]. Levels of LTB4 were not elevated during bacterial pneumonia when compared to PCP patients and healthy controls. Furthermore, a positive correlation was found between IL-8 levels in BAL fluid and relative BAL neutrophilia (r = 0.60, P = 0.001) in bacterial pneumonia. In conclusion, elevated IL-8 levels in BAL fluid were found in patients suffering from bacterial pneumonia, which may account for the influx of neutrophils to the lung, whereas LTB4 appears not to be an important chemotactic factor in this setting.

Independent risk of mechanical ventilation for AIDS-related Pneumocystis carinii pneumonia associated with bronchoalveolar lavage neutrophilia.

The use of mechanical ventilation (MV) for AIDS-related Pneumocystis carinii pneumonia (PCP) has varied over time. The introduction of adjunctive corticosteroid therapy has changed the pathophysiology of PCP. In the present study, we attempted to identify factors predictive of severe respiratory failure requiring MV amongst patients with PCP treated in the era of adjunctive corticosteroid therapy. Furthermore, we studied factors associated with survival in relation to MV. Of 170 consecutive patients with AIDS-related PCP, 18 (11%) required MV. Thirteen of 18 ventilated patients died (72%). In a logistic regression analysis, higher age, increased bronchoalveolar lavage (BAL) neutrophilia and a positive BAL cytomegalovirus CMV culture were associated with the need of MV. In multivariate analyses, only BAL neutrophilia remained independently predictive of mechanical ventilation. In conclusion, short-term mortality remained high after the introduction of adjunctive corticosteroid therapy. BAL neutrophilia may be a useful prognostic marker to identify patients at high risk of requiring mechanical ventilation.

Pneumocystis carinii pneumonia in HIV-infected patients: effect of steroid therapy on surfactant level.

Previous studies have suggested alterations in pulmonary surfactant lipid in the setting of Pneumocystis carinii pneumonia in HIV-infected patients. Because pulmonary surfactant lipid is composed of a variety of lipid products and because other phospholipids might be present in bronchoalveolar lavage (BAL) lipid determinations, a single molecular species of phospholipid which comprises a substantial portion of the surfactant lipid fraction, dipalmitoyl phosphatidylcholine (DPPC), was measured by capillary column gas chromatography in BAL samples taken at the time of the diagnosis of P. carinii pneumonia, and 10 days after treatment for P. carinii pneumonia. DPPC was measured at day 0 and day 10 in seven patients who had been randomized to receive methylprednisolone adjuvant therapy for P. carinii pneumonia and in six patients who had been randomized to not receive methylprednisolone therapy. The level of DPPC in BAL from all patients at day 0 was 0.49 +/- 0.06 microgram ml-1 BAL. This level is significantly lower that the level of DPPC determined in BAL from five normal volunteers 2.48 +/- 0.40 micrograms ml-1. At day 0, the BAL level of DPPC in patients treated with methylprednisolone was not different from the BAL level of DPPC in patients not treated with methylprednisolone. By day 10 of therapy for P. carinii pneumonia, BAL levels of DPPC in all patients had increased to 1.05 +/- 0.19 micrograms ml-1 BAL. At day 10 DPPC levels in the methylprednisolone treated group were not different from the group not treated with methylprednisolone. We conclude that in HIV-infected patients, lung surfactant lipid is reduced in the setting of P. carinii pneumonia. The lipid levels return toward normal levels with treatment. Adjuvant therapy with corticosteroids does not alter the rate of recovery of surfactant lipid levels at least after 10 days of therapy.

Interleukin-8 and eicosanoid production in the lung during moderate to severe Pneumocystis carinii pneumonia in AIDS: a role of interleukin-8 in the pathogenesis of P. carinii pneumonia.

Pneumocystis carinii pneumonia (PCP) may cause severe respiratory distress. This is believed to be partly caused by the accumulation of neutrophils in the lung. Interleukin-8 (IL-8) and leukotriene B4 (LTB4) are potent neutrophil chemo-attractants and activators. Eicosanoids [i.e. prostaglandins (PG) and leukotrienes (LT)] are pro-inflammatory mediators released from arachidonic acid by action of phospholipase A2 (PLA2) and have been implicated in the host response to micro-organisms. Bronchoalveolar lavage (BAL) was performed on patients with PCP as part of a randomized study of adjuvant corticosteroids vs. placebo, in addition to standard antimicrobial therapy. Re-bronchoscopy was offered at day 10. BAL fluid was available for 26 patients who had follow-up bronchoscopy performed. At diagnosis, IL-8 levels were elevated in patients with PCP, compared to healthy controls, and correlated with relative BAL neutrophilia and P(A-a)O2. LTB4 was also elevated in PCP, but failed to correlate with either BAL neutrophilia or P(A-a)O2. PLA2 activity in patients correlated with IL-8 levels and BAL neutrophilia, but not with P(A-a)O2. A trend towards a decrease in IL-8 levels in BAL fluid was detected in the corticosteroid-treated patients from days 0-10, whereas no change was detected in the placebo group. No change in levels of LTB4, LTC4, PGE2, PGF2a and PLA2 were detected cover time in either treatment group. This study establishes a correlation between IL-8, BAL neutrophilia and P(A-a)O2, and suggests a role of IL-8 as a mediator in the pathogenesis of PCP, whereas the role of eicosanoids seems less clear.

[Pneumocystis carinii pneumonia in HIV-infected patients examined by bronchoscopy during the period 1989-1991]. / Pneumocystis carinii pneumoni hos HIV-smittede udredt med bronkoskopi i perioden 1989-1991.

We analyzed prospectively collected data from 211 HIV-infected patients who underwent bronchoscopy because of pulmonary symptoms during the period 1989-1991. We found an improvement in survival for patients diagnosed with Pneumocystis carinii pneumonia (PCP) in 1991 as compared to 1989 and 1990 (log rank test: p = 0.07). A significant decline in PO2 and CD4 cell count was observed in patients with PCP in the same period (p < 0.05 and 0.005 respectively). We suggest that the decline in CD4 cell count and in PO2 may reflect a change in Pneumocystis carinii transmission, and that the improved survival may be a result of altered treatment strategy of PCP.

[Prognostic value of interleukin-8 in AIDS-related Pneumocystis carinii pneumonia]. / Prognostisk vaerdi af interleukin-8 ved AIDS-relateret Pneumocystis carinii-pneumoni.

UNLABELLED: We evaluated the significance of interleukin-8 (IL-8) in Pneumocystis carinii pneumonia (PCP). Bronchoalveolar lavage (BAL) fluid and serum was prospectively collected in 76 consecutive HIV-infected patients with a primary episode of PCP, as well as in ten healthy control subjects. Patients were found to have elevated levels of IL-8 in BAL fluid compared to control subjects (p < 0.01). Nine patients died during the course of PCP. Non-survivors had significantly higher IL-8 levels in BAL fluid than survivors (p < 0.05). Furthermore patients with levels of IL-8 in BAL greater then 90 pg/ml (i.e. greater than control subjects) had significantly worse vital prognosis (log rank test, p < 0.05). Thirteen patients required mechanical ventilation (MV), and these patients had significantly elevated levels of IL-8 compared with patients not requiring MV (p < 0.05). IN CONCLUSION: i) IL-8 in BAL fluid correlates to the clinical severity of the pneumonia, and ii) is a predictor of mortality and severe respiratory compromise.

Complete histological regression of Kaposi's sarcoma following treatment with protease inhibitors despite persistence of HHV-8 in lesions.

There is no current curative treatment for HIV-related Kaposi's sarcoma. The identification of human herpesvirus-8 as a possible aetiological agent suggests potential efficacy of anti-viral agents. We report here on the complete histological remission of Kaposi's sarcoma following treatment with protease inhibitors, even in patients with limited virological response and persistence of HHV-8.

Human herpes virus-8 DNA in bronchoalveolar lavage samples from patients with AIDS-associated pulmonary Kaposi's sarcoma.

Kaposi's sarcoma (KS) is the most frequent AIDS-associated neoplasm, and often disseminates to visceral organs, including the lungs. An ante-mortem diagnosis of pulmonary KS is difficult. Recently, DNA sequences resembling a new human herpes virus (HHV-8), have been identified in various forms of KS. We hypothesized that these sequences are present in samples obtained by bronchoalveolar lavage (BAL) in patients with pulmonary KS. Utilizing a nested polymerase chain reaction (PCR), 7/12 BAL cell samples from HIV-infected patients with endobronchial KS were positive for HHV-8 DNA. In contrast, only 2/39 samples from HIV-infected patients without evidence of KS were positive (p = 0.007). Detection of HHV-8 in BAL cells of patients with pulmonary KS was highly specific (95%), with a sensitivity of 58% and a positive predictive value of 78%. In conclusion, HHV-8 is associated with pulmonary KS, and PCR amplification of HHV-8 in BAL cells provides a non-invasive method with a high positive predictive value.

Neopterin and interleukin-8--prognosis in alcohol-induced cirrhosis.

BACKGROUND: Neutrophil cytotoxity and activated macrophages have been implicated in the pathogenesis of alcohol-induced liver disease. The aim of this study was to relate plasma levels of neopterin, a marker of activation of the cellular immune system, and IL-8, a neutrophil chemotactic factor, with severity of liver disease and prognosis in patients with alcohol-induced cirrhosis. METHODS: Plasma concentrations of neopterin and IL-8 were assessed in 81 patients with alcohol-induced cirrhosis admitted to the Department of Medicine B, Bispebjerg Hospital, Copenhagen, Denmark, and in 16 healthy controls. After a median follow-up period of 5 years, mortality and death causes were registered. The patients were divided into groups according to the major contributing cause of death: infection, upper gastrointestinal bleeding or hepatic coma. RESULTS: Neopterin and IL-8 levels were increased in the cirrhosis patients, but not significantly related to Child-Pugh classification. Five-year mortality was 67%. High neopterin levels (>upper quartile) were an independent predictor of death (p=0.01, Log rank and p<0.02, Cox). High IL-8 levels (>upper quartile) were of no significant prognostic value for overall mortality. Causes of death related mortality were as follows (Log rank): Neopterin; p=0.009, p=0.84 and p=0.94, and IL-8; p=0.36, p=0.002 and p=0.27, respectively, according to infection, bleeding and coma as causes of death. CONCLUSIONS: Neopterin and IL-8 plasma levels are raised in patients with alcohol-induced cirrhosis, and are predictive of mortality associated with infections and upper gastrointestinal bleeding, respectively.

Interleukin-8 in cerebrospinal fluid from patients with septic and aseptic meningitis.

Using a monoclonal antibody enzyme immunoassay, the concentration of interleukin-8 (IL-8) in cerebrospinal fluid (CSF) from 52 patients suspected of having meningitis was studied. The CSF IL-8 concentration was significantly higher in septic meningitis of known and unknown etiology than in aseptic meningitis and significantly higher in aseptic meningitis than in patients without meningitis. The CSF levels of IL-8 correlated with the levels of tumor necrosis factor-alpha, leukocyte count, neutrophil count, protein level, CSF/blood glucose ratio, and the number of days patients were hospitalized. The IL-8 values used to distinguish septic from aseptic meningitis, at a cut-off point of 3.00 micrograms/l, showed a sensitivity of 81%, a specificity of 92%, and a positive predictive value of 96%. The results suggest that determining IL-8 levels may be useful in the differential diagnosis of meningitis.

Neutrophil chemotactic activity in bronchoalveolar lavage fluid of patients with AIDS-associated Pneumocystis carinii pneumonia.

Pneumocystis carinii pneumonia (PCP) is accompanied by an acute inflammatory infiltration of the lung parenchyma. The cellular infiltrate is characterized by inflammatory cells including neutrophils, lymphocytes and macrophages. Furthermore, neutrophilia in bronchoalveolar lavage (BAL) fluid has been shown to confer a poor prognosis in PCP. We therefore investigated the potential of BAL fluid from 17 patients with PCP to induce neutrophil chemotaxis. BAL fluid from patients induced considerable neutrophil chemotactic activity compared to normal controls. Elevated levels of IL-8 were detected in patient samples as compared to controls. A specific anti-IL-8 antibody significantly reduced chemotactic activity of patient samples by more than 50%. In conclusion, IL-8 appears to be a significant participant of neutrophil chemotaxis in AIDS-associated PCP, and may participate in the recruitment of neutrophils to the lung during PCP.

Rapid loss of specific antibodies after pneumococcal vaccination in patients with human immunodeficiency virus-1 infection.

Pneumococcal infections are frequently observed in patients with human immunodeficiency virus (HIV) infection and active immunization has been recommended as prophylaxis in this patient group. We studied 103 out-patients with asymptomatic or mildly symptomatic HIV infection with respect to specific IgG and IgG2 pneumococcal antibodies before and after vaccination with a 23-valent pneumococcal polysaccharide vaccine. A significant increase ( > 2-fold) in IgG and IgG2 antibody levels was observed after 1 month in 69/103 patients (67%) with no correlation with the CD4 cell count at the time of vaccination. The response rate was not influenced by concurrent treatment with anti-retroviral monotherapy, or by age or gender. After immunization a strong correlation between IgG and IgG2 anti-pneumococcal antibodies was demonstrated. Nevertheless, 12 months after vaccination the specific antibody titres were not significantly different from pre-vaccination values. In conclusion, antibodies induced by pneumococcal vaccination in patients with HIV infection have a short duration. This raises the question as to whether vaccination will have any impact on clinical end-point in this group of patients.

Pneumocystis carinii major surface glycoprotein induces interleukin-8 and monocyte chemoattractant protein-1 release from a human alveolar epithelial cell line.

BACKGROUND: The major surface glycoprotein (MSG) is an abundant, immunogenic glycoprotein located on the surface of Pneumocystis carinii. Little is known about the proinflammatory effects of MSG. DESIGN: We have investigated the effect of human MSG on the secretion of the chemokines interleukin 8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) from an alveolar epithelial cell line (A549). RESULTS: Incubation of A549 cells with MSG in concentrations from 0.4 to 10 microg mL-1 for 24 h caused dose-dependent increases in IL-8 release (3.4-fold above control, P < 0.01). Time course experiments showed increases in IL-8 release at 4 h, 8 h and 24 h compared with control cultures (all P < 0.01). There was a minor (13%) dose- and time-related increase in MCP-1 release at 24 h (P = 0.02). Co-incubation of MSG with mannan or beta-glucan decreased IL-8 release by 48% and 42% respectively, suggesting that MSG stimulates A549 cells in part through carbohydrate moieties. Dexamethasone significantly inhibited MSG-induced IL-8 release in concentrations of 10-6-10-8 mol L-1 compared with control experiments (P < 0.01). Ribonuclease protection assays for steady-state IL-8 mRNA showed that increases in response to MSG stimulation occurred by 4 h and persisted throughout 8 h of stimulation. CONCLUSION: These findings suggest that MSG can alter alveolar epithelial cytokine release and may be capable of modulating the local inflammatory response in this manner.

Chemokine receptor CCR2b 64I polymorphism and its relation to CD4 T-cell counts and disease progression in a Danish cohort of HIV-infected individuals. Copenhagen AIDS cohort.

We have investigated the role of the recently described mutation in CCR2b named 64I in relation to HIV resistance, CD4 T-cell counts, and disease progression in Danish individuals by polymerase chain reaction (PCR)-based methods as well as sequenced full-length CXCR4 and CCR5 genes from HIV-infected long-term nonprogressors for possible mutations. In total, 215 Danish individuals were analyzed for 64I allele frequency; disease progression was followed in 105 HIV-1-positive homosexual Danish men from their first known positive HIV-1 test result and up to 11 years. In 87 individuals, the CD4 T-cell count was monitored closely. We found no significant difference in 64I allele frequency between HIV-1-seropositive persons (0.08), high-risk HIV-1-seronegative persons (0.11), and blood donors (0.06). No significant difference was observed in annual CD4 T-cell decline, CD4 T-cell counts at the time of AIDS, in AIDS-free survival as well as survival with AIDS, between 64I allele carriers and wild-type individuals. Among 9 long-term nonprogressors, 2 carried the 64I allele, while none of 9 fast progressors carried the 64I allele. However, this was not significantly different (p=.47). Long-term nonprogression could not be explained by CXCR4 polymorphism or other polymorphisms in the CCR5 gene than the CCR5delta32 allele. Furthermore, we were not able to detect any significant independent effect of the 64I allele on development to AIDS, overall survival, and annual CD4 T-cell decline in this cohort.