Anna Nanetti

Performance Assessment of New Multiplex Probe Assay for Identification of Mycobacteria

ABSTRACT A new DNA probe assay (INNO LiPA Mycobacteria; Innogenetics, Ghent, Belgium) for the simultaneous identification, by means of reverse hybridization and line-probe technology, of Mycobacterium tuberculosis complex, Mycobacterium kansasii,Mycobacterium xenopi, Mycobacterium gordonae, the species of the Mycobacterium avium complex (MAC),Mycobacterium scrofulaceum, and Mycobacterium chelonae was evaluated on a panel of 238 strains including, besides representatives of all the taxa identifiable by the system, a number of other mycobacteria, some of which are known to be problematic with the only other commercial DNA probe system (AccuProbe; Gen-Probe, San Diego, Calif.), and two nocardiae. The new kit, which includes a control probe reacting with the whole genus Mycobacterium, correctly identified 99.6% of the strains tested; the one discrepancy, which remained unresolved, concerned an isolate identified as MAC intermediate by INNO LiPA Mycobacteria and as Mycobacterium intracellulare by AccuProbe. In five cases, because of an imperfect checking of hybridization temperature, a very slight, nonspecific, line was visible which was no longer evident when the test was repeated. Two strains whose DNA failed amplification at the first attempt were regularly identified when the test was repeated. Interestingly, the novel kit dodged all the pitfalls presented by the strains giving anomalous reactions with AccuProbe. A unique feature of INNO LiPA Mycobacteria is its ability to recognize different subgroups within the species M. kansasii and M. chelonae, while the declared overlapping reactivity of probe 4 with some M. kansasii and Mycobacterium gastri organisms and of probe 9 with MAC, Mycobacterium haemophilum, andMycobacterium malmoense, may furnish a useful aid for their identification. The turnaround time of the method is approximately 6 h, including a preliminary PCR amplification.

Pseudomonas spp. complications in patients with HIV disease: An eight-year clinical and microbiological survey

Two hundred and twenty-four episodes of Pseudomonas spp. complications that occurred in 179 consecutive patients with HIV infection were retrospectively reviewed. Pseudomonas spp. organisms were responsible for 11.6% of 1933 episodes of non-mycobacterial bacterial diseases (5.4% of 1072 episodes of sepsis), observed over an 8-year period; 20.7% of patients experienced disease relapses (45 episodes). These complications mostly involved lower airways (66 cases), urinary tract (53 episodes), and blood (34 cases), with Pseudomonas aeruginosa isolated in 161 episodes, and other Pseudomonas spp. in the remaining 63 cases. An advanced HIV disease was frequently present (as expressed by a prior diagnosis of AIDS, a low CD4+ lymphocyte count, and leukopenia–neutropenia). Indwelling intravascular and urinary catheters were often associated with bacteremia and urinary tract involvement, respectively. More than 60% of patients were given antibiotics and/or cotrimoxazole in the month preceding the onset of Pseudomonas spp. disease. Bacterial strains isolated from our HIV-infected patients showed a favorable sensitivity to piperacillin, ceftazidime, imipenem, amikacin, tobramycin, and ciprofloxacin. An adequate antimicrobial treatment led to clinical and microbiological cure in 73.2% of patients at the first episode, and in 22.3% more subjects after one or more relapses. A lethal outcome occurred in only eight patients of 179 (4.5%), suffering from a far advanced HIV disease; P. aeruginosa infection directly contributed to death in four cases (sepsis, and/or pneumonia). Nosocomial disease occurred in 46.4% of the 224 episodes, and was significantly related to a previous diagnosis of AIDS, concurrent neutropenia, the occurrence of sepsis or urinary tract infection, disease relapses, the involvement of non-aeruginosa Pseudomonas spp., and a lethal outcome, compared with community-acquired infection. Our experience (the largest reported to date) confirms that Pseudomonas spp. (including non-aeruginosa Pseudomonas spp. organisms) is responsible for remarkable morbidity and mortality among patients with HIV infection, and may pose relevant problems to clinicians and microbiologists involved in the care of HIV-infected patients.

Histoplasmosis capsulati and duboisii in Europe: The impact of the HIV pandemic, travel and immigration

The present report describes a fatal case of imported AIDS-related disseminated histoplasmosis capsulati infection associated with multiple coexisting infections, diagnosed with cultural recovery ofHistoplasma capsulation var.capsulatum with a commercial radiometricMycobacterium medium. The epidemiological and clinical features of histoplasmosis capsulati and duboisii in Europe are reviewed by examining also 69 documented cases ofHistoplasma capsulatum var.capsulatum infection (25 in AIDS patients) and 17 cases ofHistoplasma capsulatum var.duboisii infection (3 in HIV-infected patients), described since 1980. This draws special attention to the role played during recent years by the emergence of the HIV pandemic and the progressive intensification of travel and immigration as risk factors for this disease in our continent. AIDS patients, who are prone to multiple concurrent opportunistic infections which may share clinical and laboratory features with each other and with other HIV-associated diseases, represent the most relevant current group at risk for severe disseminated histoplasmosis, which may come to medical attention far from their place of origin.

Fiavobacteriurrt spp. organisms as opportunistic bacterial pathogens during advanced HIV disease

OBJECTIVE To assess the role of Flavobacterium spp. infection in patients with HIV disease. METHODS Clinical charts of 2412 consecutive HIV-infected patients hospitalized in a 8-year period were retrospectively reviewed, to identify all cases of Flavobacterium spp. infections, and to evaluate their occurrence and outcome according to several epidemiological, clinical, and laboratory parameters. RESULTS Six patients out of 2412 (0.25%), developed Flavobacterium spp. complications: septicaemia in five cases, and pneumonia in the remaining patient, with F. meningosepticum and F. odoratum isolated in two cases and one case, respectively, and unnamed Flavobacterium spp. organisms in the remaining three cases. Flavobacterium spp. organisms were responsible for six out of 1939 overall episodes of non-mycobacterial bacterial diseases observed in our patient group (0.31%). All patients were severely immunocompromised, showing a prior diagnosis of AIDS, a mean CD4+ lymphocyte count of 64.2 (range 12-187) cells/microl, and a mean neutrophil count of 1.143 (range 700-1600) cells (range 700-1600) cells/microl. Antibiotic, corticosteriod, or cotrimoxazole treatment was carried out during the month preceding disease onset by three, two and five patients, respectively. Community-acquired and nosocomial Flavobacterium spp. disease were equally frequent, but the latter occurred with a significantly lower mean neutrophil and CD4+ cell count. Antimicrobial susceptibility assays showed complete sensitivity to ciprofloxacin, and variable resistance to ureidopenicillins, ceftazidime, imipenem, aztreonam, and aminoglycosides. An appropriate antimicrobial regimen obtained clinical and microbiological cure in all cases, in absence of related mortality or relapses. CONCLUSIONS Since only one episode of HIV-associated F. (Sphingobacterium) multivorum complication has been described to date, our series represents the largest one dealing with Flavobacterium spp. infection in the setting of HIV disease. Our experience suggests that Flavobacterium spp. organisms may play a pathogenic role in patients with advanced HIV disease, even when some commonly recognized risk factors are lacking (i.e. indwelling catheters, instrumentation, IV drug abuse), while a very low CD4+ lymphocyte count, leukopaenia-neutropaenia, and concurrent AIDS-related infectious complications may act as important predisposing factors. In view of the infrequent occurrence of these infections, early suspicion is essential for both clinicians and microbiologists facing immunocompromised patients at risk for invasive bacterial complications. Flavobacterium spp. organisms should be taken into consideration as nosocomial- or community-acquired opportunistic pathogens, due to their relationship with advanced immunodeficiency and their elevated resistance to many antimicrobial agents commonly used against Gram-negative bacterial pathogens.

Bacteremia and respiratory involvement byAlcaligenes xylosoxidans in patients infected with the human immunodeficiency virus

Seven cases ofAlcaligenes xylosoxidans bacteremia and/or respiratory disease in patients infected with the human immunodeficiency virus (HIV) are described. Reported only thrice previously in this setting, these bacterial complications occurred during different phases of HIV infection and were associated with leukopenia-neutropenia in four patients and a central vascular catheter in two. Although the majority of cases were diagnosed after day 3 of hospitalization, a distinct source of infection was never identified. In four patients with advanced underlying disease, a polymicrobial infection was present. In vitro resistance to aminoglycosides, first-generation cephalosporins, and aztreonam was identified, but treatment with fluoroquinolones, piperacillin, or an aminoglycoside in combination with either ceftazidime or pefloxacin was successful in all cases. The relevance ofAlcaligenes xylosoxidans and related species of gramnegative non-glucose fermenting bacilli as opportunistic pathogens in the immunocompromised host and in the setting of HIV infection is briefly reviewed.

Histoplasma capsulatum var. capsulatum occurring in an HIV‐positive Ghanaian immigrant to Italy

Histoplasmosis, which is highly endemic in the United States, is rare in Europe, usually imported but sometimes autochthonous. In Africa, histoplasmosis capsulati coexists with “African histoplasmosis”, a characteristic skin infection caused by H. capsulatum var. duboisii. Histoplamosis due to H. capsulatum is one of the 12 secondary infections listed in the surveillance definitions of AIDS. We report the case of a 36‐year‐old black man with acquired immunodeficiency syndrome (AIDS) who was living in Italy but originally came from Ghana. Histoplasmosis was disseminated with fever and cutaneous manifestations. The diagnosis was demonstrated morphologically based on the presence of yeast, observed by light microscopy, in skin lesions and by identification of H. capsulatum var. capsulatum DNA by nested PCR from a paraffin sample. No clinical reports of histoplamosis capsulati in Ghana have been published until now. The present case stresses the role of immigration of subjects from outside Europe who have been infected in their native country.

Xanthomonas maltophilia : an emerging pathogen in patients with HIV disease:

Fifty-four episodes of Xanthomonas maltophilia infection were observed in 52 HIV-infected patients out of 2062 assessed (2.52%) over a 6-year period: sepsis/ bacteraemia in 44 cases, lower airways infection in 5 cases, urinary tract infection and pharyngitis in 2 cases each, and lymph node involvement in one patient. X. maltophilia represented the fourth most common non-mycobacterial bacterial pathogen responsible for bacteraemia in HIV-infected patients: 44 cases out of 721 diagnosed (6.1%). When compared with non-typhoid Salmonella spp. bacteraemia, an increased risk to develop X. maltophilia disseminated infection was seen according to the progression of HIV-related immunodeficiency, the occurrence of leukopenia-neutropenia, central venous catheterization, previous antibiotic and/or corticosteroid treatment, and hospitalization. In 3 patients suffering from concurrent AIDS-related disorders, X. maltophilia infection contributed to death, while a recurrence occurred in 2 cases only. Due to the poor antimicrobial susceptibility of this pathogen (also confirmed in our series), X. maltophilia bacteraemia associated with advanced HIV infection and concurrent risk factors, may represent a potentially severe disease.

Fatal Campylobacter jejuni bacteraemia in patients with AIDS

Two fatal cases of Campylobacter jejuni septicaemia in patients with AIDS were characterised by severe HIV-related immunodeficiency, negative stool cultures and presentation during hospitalisation, developing a clinical picture of fulminant septic shock despite therapy with appropriate antibiotics. Campylobacter spp. are important opportunist pathogens in HIV disease and may cause a septicaemic illness in the absence of enteric disease.

An outbreak of Salmonella Hadar associated with roast rabbit in a restaurant

In August 1997, an outbreak of gastroenteritis from SalmonellaHadar phage type 2 occurred among customers of a restaurant in Rimini (Emilia-Romagna region, Italy). Twenty-nine people who had eaten food prepared in the restaurant on 2 or 3 August had symptoms of acute gastroenteritis. The infection was culture-confirmed in 24 cases and the stool specimens of four healthy people were positive for SalmonellaHadar. Twelve people had to be hospitalized and a 3-year old girl died. The case–control study identified roast rabbit as the likely vehicle of infection (OR: 6.00; CI 95%: 1.65–22.83). The microbiological investigation carried out on food taken from the restaurant confirmed high levels of SalmonellaHadar in a sample of roast rabbit. Since the rabbit was well cooked, the food contamination likely occurred after cooking. Poor hygienic conditions found in the restaurant, together with inappropriate food-handling practices and inadequate storage temperatures may have contributed to spread to other foods and the severity of the outbreak.

Nosocomial Clostridium difficile-associated diarrhea in patients with AIDS: a three-year survey and review.

to treatment, with resolution of the diarrhea a mean of 7 days after A total of 985 stool samples from 312 patients were examined. antibiotic therapy was instituted. These patients were cured and During the survey period, 35 (11%) of the 312 patients with diar- remained asymptomatic during the follow-up period of 12 months. rhea were identified from the microbiology records as having cul- The drugs were well tolerated by all patients; there were no sigtures and cytotoxin assays positive for C. difficile. Twenty-six nificant adverse events. Despite initial treatment with metronidaepisodes of CDAD were identified in 19 (54%) of the 35 patients zole, five patients relapsed at a mean time of 18 days after treatment with AIDS. There were 13 men (68%) and 6 women (32%), and was discontinued. In these cases the diarrhea completely resolved the mean age was 29 years (range, 4‐43 years). AIDS was diag- after therapy with vancomycin or teicoplanin was instituted. nosed before the onset of CDAD in all cases. HIV infection was C. difficile causes a wide spectrum of disease in patients with acquired through intravenous drug abuse by eight patients (five AIDS, and CDAD may represent £12.5% of the diarrheas that males and three females), homosexual relations (five males), and occur in this patient population [1]. Although HIV infection itself heterosexual relations (three males and three females). The CD4 / is not considered an intrinsic risk factor for C. difficile infection, lymphocyte count was very low in most instances (median count, individuals infected with HIV appear to be at increased risk for 48/mm 3 ; range, 5‐115/mm 3 ). CDAD [1‐6]. The predisposing conditions for acquiring C. diffiNo cases of diarrhea were due to medications or concomitant cile disease include the use of antibiotics or antineoplastic agents, infection with protozoa, mycobacteria, viruses, or bacterial patho- gastrointestinal procedures or surgery, prolonged hospitalization, gens. Endoscopic examinations were performed for 11 patients; and severe underlying disease that may increase host susceptibility evidence of severe colitis was present in eight of these patients, to infection. Patients with AIDS frequently require recurrent and and the presence of pseudomembranous colitis could be confirmed prolonged hospitalization, leading to colonization with nosocomial by histology for three other patients. Presenting complaints in- microorganisms including C. difficile. The frequent exposure of cluded watery diarrhea (85% of patients), abdominal pain (90%), these patients to prolonged or multiple courses of antibimicrobial fever (80%), nausea (60%), weakness (65%), and bloody diarrhea or anticancer chemotherapy may result in profound changes in the composition of endogenous bacteria in the alimentary tract, leading to the overgrowth of toxigenic C. difficile. A variety of antimicrobial agents have been linked with the