New vaccines for the prevention of tuberculosis in human immunodeficiency virus infection.

The prevention of human immunodeficiency virus (HIV) associated tuberculosis (TB) remains challenging. Several vaccines against TB have advanced to clinical trials in patients with HIV infection. The DarDar Trial, a large, randomized, placebo-controlled efficacy trial conducted in Tanzania, has demonstrated that a multiple dose series of an inactivated whole cell mycobacterial vaccine is safe in HIV and can prevent HIV-associated TB in patients with childhood bacille Calmette-Guérin vaccination and CD4 counts of ≥200 cells/mm(3). These developments offer promise that in the not too distant future immunization with an effective vaccine against TB can be added to other strategies for the prevention of HIV-associated TB. This expanded approach is referred to as the Five 'I's': intensified case finding, infection control, isoniazid preventive therapy (IPT), initiation of antiretroviral therapy (ART), and immunization against TB. We encourage additional studies of new TB vaccines in HIV, and propose a strategy to reduce the risk of TB by integrating IPT, ART and immunization into routine HIV care. At the time of HIV diagnosis, patients with CD4 counts of ≥200 cells/mm(3) could receive immunization, IPT and, as appropriate, ART. In patients presenting with lower CD4 counts or already on ART, immunization could be initiated at CD4 counts of ≥200 cells/mm(3) to add to the protection afforded by IPT and ART.

Completion of isoniazid preventive therapy and survival in HIV-infected, TST-positive adults in Tanzania.

SETTING: The World Health Organization recommends the use of isoniazid preventive therapy (IPT) for human immunodeficiency virus (HIV) infected patients with a positive tuberculin skin test (TST). However, due to concerns about the effectiveness of IPT in community health care settings and the development of drug resistance, these recommendations have not been widely implemented in countries where tuberculosis (TB) and HIV co-infection is common. OBJECTIVE: To evaluate the effectiveness of IPT on survival and TB incidence among HIV-infected patients in Tanzania. DESIGN: A cohort study nested within a randomized trial of HIV-infected adults with baseline CD4 counts of ≥ 200 cells/µ l was conducted to compare survival and incidence of active TB between TST-positive subjects who did or did not complete 6 months of IPT in the period 2001-2008. RESULTS: Of 558 TST-positive subjects in the analytic cohort, 488 completed 6 months of IPT and 70 did not. Completers had a decrease in mortality compared to non-completers (HR 0.4, 95%CI 0.2-0.8). However, the protective effect of IPT on the incidence of active TB was non-significant (HR 0.6, 95%CI 0.3-1.3). CONCLUSION: Completion of IPT is associated with increased survival in HIV-infected adults with CD4 counts ≥ 200 cells/µ l and a positive TST.

Disseminated tuberculosis in human immunodeficiency virus infection: ineffective immunity, polyclonal disease and high mortality.

BACKGROUND: Disseminated tuberculosis (TB) is a major cause of death in patients with the acquired immune-deficiency syndrome (AIDS), but its pathogenesis and clinical features have not been defined prospectively. METHODS: Human immunodeficiency virus (HIV) infected adults with a CD4 count ≥ 200 cells/µl and bacille Calmette-Guérin scar underwent immunologic evaluation and subsequent follow-up. RESULTS: Among 20 subjects who developed disseminated TB, baseline tuberculin skin tests were ≥15 mm in 14 (70%) and lymphocyte proliferative responses to Mycobacterium tuberculosis were positive in 14 (70%). At the time of diagnosis, fever ≥2 weeks plus ≥5 kg weight loss was reported in 16 (80%) patients, abnormal chest X-rays in 7/17 (41%), and positive sputum cultures in 10 (50%); median CD4 count was 30 cells/µl (range 1-122). By insertion sequence (IS) 6110 analysis, 14 (70%) blood isolates were clustered and 3/8 (37%) concurrent sputum isolates represented a different strain (polyclonal disease). Empiric TB treatment was given to eight (40%) patients; 11 (55%) died within a month. CONCLUSIONS: Disseminated TB in HIV occurs with cellular immune responses indicating prior mycobacterial infection, and IS6110 analysis suggests an often lethal combination of reactivation and newly acquired infection. Control will require effective prevention of both remotely and recently acquired infection, and wider use of empiric therapy in patients with advanced AIDS and prolonged fever.

A vaccine to prevent herpes zoster and postherpetic neuralgia in older adults.

BACKGROUND: The incidence and severity of herpes zoster and postherpetic neuralgia increase with age in association with a progressive decline in cell-mediated immunity to varicella-zoster virus (VZV). We tested the hypothesis that vaccination against VZV would decrease the incidence, severity, or both of herpes zoster and postherpetic neuralgia among older adults. METHODS: We enrolled 38,546 adults 60 years of age or older in a randomized, double-blind, placebo-controlled trial of an investigational live attenuated Oka/Merck VZV vaccine ("zoster vaccine"). Herpes zoster was diagnosed according to clinical and laboratory criteria. The pain and discomfort associated with herpes zoster were measured repeatedly for six months. The primary end point was the burden of illness due to herpes zoster, a measure affected by the incidence, severity, and duration of the associated pain and discomfort. The secondary end point was the incidence of postherpetic neuralgia. RESULTS: More than 95 percent of the subjects continued in the study to its completion, with a median of 3.12 years of surveillance for herpes zoster. A total of 957 confirmed cases of herpes zoster (315 among vaccine recipients and 642 among placebo recipients) and 107 cases of postherpetic neuralgia (27 among vaccine recipients and 80 among placebo recipients) were included in the efficacy analysis. The use of the zoster vaccine reduced the burden of illness due to herpes zoster by 61.1 percent (P<0.001), reduced the incidence of postherpetic neuralgia by 66.5 percent (P<0.001), and reduced the incidence of herpes zoster by 51.3 percent (P<0.001). Reactions at the injection site were more frequent among vaccine recipients but were generally mild. CONCLUSIONS: The zoster vaccine markedly reduced morbidity from herpes zoster and postherpetic neuralgia among older adults.

Sources of disseminated Mycobacterium avium infection in AIDS.

OBJECTIVES: To identify the sources of disseminated Mycobacterium avium complex (MAC) infection in AIDS. METHODS: HIV positive subjects with CD4 counts <100/mm(3) in Atlanta, Boston, New Hampshire and Finland were entered in a prospective cohort study. Subjects were interviewed about potential MAC exposures, had phlebotomy performed for determination of antibody to mycobacterial lipoarabinomannin and for culture. Patient-directed water samples were collected from places of residence, work and recreation. Patients were followed for the development of disseminated MAC. Univariate and multivariate risk factors for MAC were analyzed. RESULTS: Disseminated MAC was identified in 31 (9%) subjects. Significant risks in univariate analysis included prior Pneumocystis carinii pneumonia (PCP) (hazard ratio 1.821), consumption of spring water (4.909), consumption of raw seafood (34.3), gastrointestinal endoscopy (2.894), and showering outside the home (0.388). PCP, showering and endoscopy remained significant in a Cox proportional hazards model. There was no association between M. avium colonization of home water and risk of MAC. In patients with CD4<25, median OD antibody levels to lipoarabinomannin at baseline were 0.054 among patients who did not develop MAC and 0.021 among patients who did develop MAC (P=0.077). CONCLUSIONS: MAC infection results from diverse and likely undetectable environmental and nosocomial exposures. Mycobacterial infection before HIV infection may confer protection against disseminated MAC in advanced AIDS.

Molecular strain typing of Campylobacter jejuni by pulsed-field gel electrophoresis in a single day.

Rapid molecular strain typing is critical for effective outbreak investigation and implementation of infection control measures. Pulsed-field gel electrophoresis is a highly discriminatory technique for Campylobacter jejuni, but generally requires 3-5 days. We describe a simplified protocol for pulsed-field gel electrophoresis that provides high quality typing of C. jejuni isolates in a single day.

Phyloproteomics: species identification of Enterobacteriaceae using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

To evaluate matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) as a tool for rapid identification of common clinical bacterial isolates, we analyzed 25 carefully selected isolates of pathogenic Escherichia coli (E. coli) and additional Enterobacteriaceae members. Organisms were prepared according to clinical microbiological protocols and analyzed with minimal additional processing. Spectra were reproducible from preparation to preparation and comprised 40-100 peaks primarily representing intracellular proteins with masses up to 25 kDa. Spectra of 14 genetically diverse bacteremic isolates of E. coli were compared with isolates representing other genera within the Enterobacteriaceae family. Using a new spectrum comparison algorithm, E. coli isolates were closely related to each other and were readily distinguishable from other Enterobacteriaceae, including Salmonella and Shigella. Presently, the methodology permits the analysis of 40 unknown isolates per hour per instrument. These results suggest that MALDI-ToF MS offers a rapid and reliable approach for performing phyloproteomics i.e., identification of unknown bacterial isolates based on similarities within protein biomarker databases.

Identification of Mycobacterium avium DNA sequences that encode exported proteins by using phoA gene fusions.

SETTING: Mycobacterium avium is the major cause of disseminated infection in patients with late stage AIDS. OBJECTIVE: In order to identify M. avium genes that may be involved in bacterial uptake and intracellular survival, a phoA -based reporter system was used to identify genes that encoded surface-expressed or exported proteins. DESIGN: PhoA (alkaline phosphatase) is only active if the protein is exported across the cell membrane into the periplasm. Consequently, detectable PhoA activity requires the fusion of a promoterless phoA gene with a DNA fragment containing a functional promoter and export leader sequence. A M. avium promoter library was constructed in the phoA reporter plasmid pJEM11 and screened in M. smegmatis for expression of active PhoA. RESULTS: More than 100 independent PhoA(+)recombinants were isolated, of which 15 were sequenced. Most of these exhibited varying degrees of homology with published M. avium, M. tuberculosis, M. bovis and M. leprae sequences. Based on sequence homology, one M. avium sequence was identified as a homologue of the M. tuberculosis phosphate transport gene phoS2 (Ag88). Another M. avium sequence was homolog with a putative M. tuberculosis cutinase gene. Both of these M. avium genes were cloned and sequenced. Several other M. avium sequences were homologous with, as yet, unidentified M. tuberculosis genes. CONCLUSION: PhoA fusion technology is applicable to the study of atypical slow growing mycobacteria. Most of the M. avium exported proteins identified in this study are highly homologous with genes from M. tuberculosis and M. leprae. In addition, parallels in gene organization were identified between M. avium and members of the M. tuberculosis complex.

A macrophage invasion mechanism for mycobacteria implicating the extracellular domain of CD43.

We studied the role of CD43 (leukosialin/sialophorin), the negatively charged sialoglycoprotein of leukocytes, in the binding of mycobacteria to host cells. CD43-transfected HeLa cells bound Mycobacterium avium, but not Salmonella typhimurium or Shigella flexneri. Quantitative bacteriology showed that macrophages (M(phi)) from wild-type mice (CD43(+/+)) bound M. avium, Mycobacterium bovis (bacillus Calmette-Guérin), and Mycobacterium tuberculosis (strain H37Rv), whereas M(phi) from CD43 knockout mice (CD43(-/)-) did not. Fluorescence microscopy demonstrated that the associated M. avium had been ingested by the CD43(+/+) M(phi). The inability of CD43(-/)- M(phi) to bind M. avium could be restored by addition of galactoglycoprotein (Galgp), the extracellular mucin portion of CD43. The effect of Galgp is not due to opsonization of the bacteria, but required its interaction with the M(phi) other mucins had no effect. CD43 expression by the M(phi) was also required for optimal induction by M. avium of tumor necrosis factor (TNF)-alpha production, which likewise could be reconstituted by Galgp. In contrast, interleukin (IL)-10 production by M. avium-infected M(phi) was CD43 independent, demonstrating discordant regulation of TNF-alpha and IL-10. These findings describe a novel role of CD43 in promoting stable interaction of mycobacteria with receptors on the M(phi) enabling the cells to respond specifically with TNF-alpha production.

Safety and immunogenicity of a five-dose series of inactivated Mycobacterium vaccae vaccination for the prevention of HIV-associated tuberculosis.

Five doses of inactivated Mycobacterium vaccae vaccine were administered intradermally to 22 human immunodeficiency virus (HIV)-infected patients (11 bacille Calmette-Guérin [BCG]-positive and 11 BCG-negative) in Zambia whose CD4 lymphocyte counts were >/=200 cells/mm(3). HIV viral load and lymphocyte proliferation responses were compared for vaccine recipients and 22 HIV-infected control patients (11 BCG-positive and 11 BCG-negative). Immunization was safe and well tolerated in all patients, and induration at the vaccine site decreased from dose 1 to dose 5. A transient decrease in HIV viral load was observed in BCG-positive vaccine recipients after dose 3 but not after subsequent doses. Median lymphocyte stimulation indices to M. vaccae were 6.0 in vaccine recipients and 2.3 in control patients (P<.001). Stimulation indices were >/=3.0 in 19 vaccine recipients (86%) and 7 control patients (32%; P=.001). A 5-dose series of vaccination with inactivated M. vaccae is safe in HIV-infected patients and induces lymphocyte proliferation responses to the vaccine antigen. M. vaccae vaccine is a candidate for the prevention of tuberculosis in HIV infection.

Macrophage apoptosis in mycobacterial infections.

Mycobacterial diseases are a major public health concern. In the case of tuberculosis, the problem has been acerbated due to the emergence of drug-resistant strains of Mycobacterium tuberculosis, and Mycobacterium avium is the major opportunistic pathogen in HIV-1 infection in the United States. M. tuberculosis and M. avium replicate in human macrophages and induce apoptosis. Incubation of freshly added uninfected autologous macrophages with apoptotic M. avium-infected macrophages results in 90% inhibition of bacterial growth. Apoptosis also prevents the release of intracellular components and the spread of mycobacterial infection by sequestering the pathogens within apoptotic bodies. Consistent with the model that host cell apoptosis is a defense mechanism against mycobacteria is the finding that the virulent M. tuberculosis strain H37Rv induces substantially less macrophage apoptosis than the attenuated strain H37Ra. Evasion of apoptosis by this pathogen is achieved by enhanced release of sTNFR2 by H37Rv-infected macrophages and subsequent formation of inactive TNF-alpha-TNFR2 complexes. These observations contribute to the hypothesis that apoptosis of the host macrophage is an important defense mechanism in mycobacterial infections, which prevents the spread of the infection.

High rates of disseminated infection due to non-tuberculous mycobacteria among AIDS patients in Finland.

OBJECTIVE: to determine the rate of disseminated infection due to non-tuberculous mycobacteria (NTM) among Finnish AIDS patients, and to analyse the epidemiology of these infections. METHODS: in a prospective cohort study HIV-infected patients with CD4 counts < 200 x 10(6)/l were interviewed, and had mycobacterial blood cultures performed at baseline and at 6 months, then subsequently for clinical indications; autopsies were performed on patients who died. The cohort was followed at least for 24 months or to death. Water samples were collected from the homes of patients and from the environment and cultured for organisms of the Myobacterium avium complex (MAC). Environmental and clinical isolates were compared using pulsed field gel electrophoresis (PFGE). RESULTS: NTM infection occurred in 22 (43%) of 51, 19 isolates were Mycobacterium avium, two M. genavense and one M. intracellulare. Multivariate analysis identified urban residence (P=0.04) and eating raw fish (P=0.04) as independent risk factors. Molecular analysis revealed two clusters of related isolates (three M. avium, two M. genavense) among urban residents. CONCLUSION: AIDS patients in Finland have high rates of disseminated infection due to NTM. Clusters of identical organisms and association with urban residence suggests that these are newly acquired infections in advanced AIDS.

Randomised trial of intradermal Mycobacterium vaccae or intradermal hepatitis B immunisation in children with HIV infection.

This study assessed the safety of inactivated Mycobacterium vaccae as a candidate vaccine to prevent disseminated mycobacterial disease in children with HIV infection. 35 children ages 1-8 with CD4 counts > or =300/mm3 in New Hampshire, Boston and Chicago were randomised in a 2:1 schedule to receive a 3-dose series of intradermal M. vaccae vaccine (MV) or hepatitis B vaccine (HBV) at 2-month intervals. Immunisation was safe and well tolerated; 2-day median vaccine site in duration was 5 mm in MV recipients and 0 mm in HBV recipients (p < 0.001). There were no significantly different changes in viral load or CD4 count between the two vaccine groups. No PPD skin test conversions occurred after immunisation. MV is safe and well tolerated and deserves further evaluation as a vaccine to prevent mycobacterial disease in HIV-infected children.

Isolation of mycobacteria from indoor swimming pools in Finland.

The presence of mycobacteria in seven indoor pools in Finland was evaluated by multiple culture methods. Replicate samples, with and without inactivation of chlorine by sodium thiosulfate, were cultured in two laboratories. Laboratory I used two methods: (A) no decontamination and (B) cetylpyridinium chloride (0.005%, 20 min); and Laboratory II two methods: (C) cetylpyridinium chloride (0.005%, 18 h) and (D) oxalic acid (5%, 15 min). Samples processed by methods (A) and (B) were cultured on different egg media of pH 6.3 or 5.8; by method (C) on Middlebrook and Cohn 7H10 (+OADC) agar of pH 5.5; and by method (D) on Middlebrook and Cohn 7H10 agar (+OADC) with cycloheximide (500 microg/ml). Mycobacteria were recovered from five (71%) of seven pools. Detection of mycobacteria depended on the method used. High isolation rates (36-46% of the samples) were obtained by methods (A), (B) and (D). Contamination was a problem only with method (A). Inactivation of chlorine had a variable impact on mycobacterial detection. Isolates included M. kansasii, M. gordonae, M. fortuitum complex, M. sphagni, and M. vaccae, as well as M. simiae-like and M. chubuense-like organisms. In addition, a group of slowly growing and a group of rapidly growing isolates with previously unknown fatty acid and alcohol composition were isolated. No M. avium was detected. Mycobacterial counts were highest in a small pool with high temperature, low pH, and low content of free available chlorine.

Nasal carriage of and infection with Staphylococcus aureus in HIV-infected patients.

BACKGROUND: Staphylococcus aureus is a common cause of serious infection in patients infected with HIV. OBJECTIVES: To evaluate risk factors for and quantitative effect of S. aureus infection in HIV-infected patients, with special attention to nasal carriage. DESIGN: Prospective, multihospital cohort study. SETTING: Three tertiary care Veterans Affairs Medical Centers. PARTICIPANTS: 231 ambulatory HIV-infected patients. RESULTS: Thirty-four percent of patients were nasal carriers of S. aureus. Of these patients, 38% were persistent carriers and 62% were intermittent carriers. Twenty-one episodes of infection occurred in 13 patients: Ten were bacteremias (including 2 cases of endocarditis), 1 was pneumonia, and 10 were cutaneous or subcutaneous infections. Seventeen (85%) of these episodes occurred in patients with CD4 counts less than 100 cells/mm3. Recurrent infections occurred in 3 of 7 patients who survived an initial S. aureus infection. The mortality rate was higher among patients with S. aureus infection than among those without infection (P = 0.03). Factors significantly associated with S. aureus infection were nasal carriage, presence of a vascular catheter, low CD4 count, and neutropenia. Molecular strain typing indicated that for 6 of 7 infected patients, the strain of S. aureus isolated from the infected sites was the same as that previously cultured from the nares. CONCLUSION: Nasal carriage is an important risk factor for S. aureus infection in HIV-infected patients. Controlled studies are indicated to determine whether eradication of nasal carriage in a selected subset of patients (for example, those with a low CD4 cell count) might prevent invasive S. aureus infection in patients with HIV infection.

Characterization of IS1245 for strain typing of Mycobacterium avium.

IS1245 is an insertion element widely prevalent among isolates of Mycobacterium avium. We used PvuII Southern blots to analyze IS1245 polymorphisms among 159 M. avium isolates (141 clinical isolates from 40 human immunodeficiency virus-infected patients plus 18 epidemiologically related environmental isolates) that represented 40 distinct M. avium strains, as resolved by previous studies by pulsed-field gel electrophoresis (PFGE). All 40 strains carried DNA homologous to IS1245 and thus were typeable. Twenty-five (63%) strains had > or = 10 copies of the element, 6 (15%) had 4 to 9 copies, and 9 (23%) had only 1 to 3 copies. Among the last group of nine strains (each of which was distinct by PFGE analysis), IS1245 typing resolved only four patterns and thus provided poor discriminatory power. To evaluate the in vivo stability of IS1245, we analyzed 32 strains for which sets of 2 to 19 epidemiologically related isolates were available. For 19 (59%) of these sets, all isolates representing the same strain had indistinguishable IS1245 patterns. Within eight (25%) sets, one or more isolates had IS1245 patterns that differed by one or two fragments from the modal pattern for the isolates of that strain. Five (16%) sets included isolates whose patterns differed by three or more fragments; on the basis of IS1245 typing those isolates would have been designated distinct strains. IS1245 was stable during in vitro passage, suggesting that the variations observed represented natural translocations of the element. IS1245 provides a useful tool for molecular strain typing of M. avium but may have limitations for analyzing strains with low copy numbers or for resolving extended epidemiologic relationships.

Sensitivity and specificity of an improved rapid latex agglutination test for identification of methicillin-sensitive and -resistant Staphylococcus aureus isolates.

The performance of a second-generation rapid agglutination kit, Slidex Staph Plus (SSP; bioMérieux), was compared to those of the Slidex Staph (SS; bioMérieux), Staphaurex (SRX; Murex Diagnostics), and BBL Staphyloslide (BBL; Becton Dickinson) kits by using 508 clinical isolates composed of 150 methicillin-sensitive Staphylococcus aureus (MSSA) organisms, 154 methicillin-resistant S. aureus (MRSA) organisms, and 204 non-S. aureus Staphylococcus spp. Of the 508 isolates tested, 75% were fresh clinical isolates, with the remainder taken from five different freezer collections. All four agglutination tests had comparable sensitivities for MSSA and MRSA. However, the SS kit was significantly less specific (93.1%) than the three other tests (P > 0.05, McNemar test). These results demonstrate that the new rapid latex agglutination kit, SSP, was more specific for the identification of S. aureus than the previous version and performed comparably to the SRX and BBL kits.

Immunization of HIV-infected adults with a three-dose series of inactivated Mycobacterium vaccae.

Heat-killed Mycobacterium vaccae vaccine was administered in a 3-dose schedule to 12 HIV-infected adults with CD4 cell counts > or = 300/mm3. Local and systemic side effects were monitored. Delayed-type hypersensitivity to purified protein derivative and Mycobacterium avium sensitin was measured at baseline and after the final dose. Antibody to aralipoarabinomannin, man-lipoarabinomannin, and a short-term culture filtrate of Mycobacterium tuberculosis were also measured. Lymphocyte proliferation responses to M avium sensitin and M vaccae sonicate were determined. Vaccine site induration was maximal at 2 days (median, 6 mm) and no systemic side effects were noted. Purified protein derivative skin test conversions did not occur. Changes in CD4 counts and HIV viral load were not significant. Three (27%) of 11 subjects who completed the trial showed either M avium skin test (n = 1) or short-term culture filtrate antibody (n = 2) responses. A three-dose schedule of M vaccae vaccine is safe and well tolerated in adults with early HIV infection and produces detectable immunologic responses in a subset of these subjects.