Viral monitoring and successful treatment of a ganciclovir-resistant cytomegalovirus infection in a heart transplant recipient.

We reported a ganciclovir (GCV)-resistant cytomegalovirus (CMV) infection in a heart transplant recipient. Genotypic and phenotypic susceptibility assays demonstrated an A594V mutation in the UL97 phosphotransferase gene and GCV IC(50)>96 microM. Low GCV concentration exposure, immunosuppressive treatment, donor-positive/recipient-negative CMV serostatus, viral reactivations within antiviral prophylaxis or treatment, contributed to GCV-resistant strain selection.

Comparison of cytomegalovirus viral load measure by real-time PCR with pp65 antigenemia for the diagnosis of cytomegalovirus disease in solid organ transplant patients.

Cytomegalovirus (CMV) infection is the most frequent complication in solid organ transplant recipients. Currently, the antigenemia assay is widely used to detect this infection, although its success is being questioned to a great extent nowadays. The aim of our study is to compare a quantitative real time PCR to measure CMV DNA to the antigenemia assay, for the diagnosis to CMV disease. For our research, we prospectively processed 1198 samples (plasma and peripheral blood leukocytes [PBMC]), which belonged to 158 transplant recipients. In every sample the detection of the pp65 antigen in PBMC was carried out, as well as the quantification of CMV DNA by PCR (Light Cycler, LC-PCR). For this process, FRET probes, which detect a 254-bp fragment from the CMV gB gene, were used. The dynamic range of the LC-PCR was 500 to 5.10(7) copies/mL plasma and from 62 to 6.10(6) copies/10(6) PBMC. Twenty-three episodes of cytomegalovirus (CMV) disease occurred in 22 out of 158 patients and PCR displayed levels of sensitivity and specificity of 100% and 67%, respectively. The antigenemia assay obtained values of 91% and 57%. We established a cutoff value of 10(3) copies/mL plasma and 315 copies/10(6) cells. According to these cutoff values, PCR showed levels of sensitivity, specificity, VPN and VPP of 95.6%, 81.6%, 99%, and 53% respectively. Moreover, the LC-PCR assay anticipated the antigenemia assay in 10 patients out of 22 who developed CMV disease and the appearance of any clinical symptoms in 12 out of 22 patients. In conclusion, we believe that the quantification of CMV DNA by LC-PCR is a superior assay to pp65 antigenemia test regarding the early diagnosis of CMV disease in solid organ transplant recipients.

A simple broth-disk elution test for screening methicillin-resistant (heteroresistant) staphylococci.

Forty-six strains of Staphylococcus aureus and 47 strains of unspeciated coagulase-negative staphylococci were inoculated in tubes containing a cation-supplemented Mueller-Hinton broth with 4% (wt/vol) NaCl. A critical concentration of methicillin (10 micrograms/ml) or oxacillin (6 micrograms/ml) was achieved in each tube by adding the appropriate number of antibiotic disks. Growth was interpreted as resistance. Results obtained with fully resistant or fully sensitive strains showed complete agreement with the reference method. Four strains with intermediate sensitivity (two S. aureus and two coagulase-negative staphylococci) were classified as resistant by the broth-disk elution test.

[Prospective study of cytomegalovirus infection in liver transplant recipients]. / Estudio prospectivo de la infección por citomegalovirus en receptores de trasplante hepático.

BACKGROUND: Cytomegalovirus (CMV) infection is an important cause of morbidity and mortality in liver transplant recipients. The aim of this study was to determine the incidence, clinical characteristics, risk factors and response to treatment of CMV infection in liver transplant recipients. METHODS: Fifty-nine adult patients who underwent a liver transplant in our hospital were prospectively studied. Cell cultures were used to document CMV infection and disease. Ganciclovir treatment was only given to patients with documented CMV disease. RESULTS: Active infection was shown in 47 of 59 patients (80%), and symptomatic infection (disease) in 17 (29%). The most frequent clinical presentation was hepatitis (9 cases) followed by flu-like syndrome (6 cases) and pneumonitis (4 cases). The average time from transplant to the diagnosis of CMV disease was 36 days. The seronegative recipients of seropositive donors developed symptomatic infection more frequently (66%) than other patients (p < 0.05). As well as the CMV serologic status before transplantation, the use of anti-OKT3 antibodies was the only risk factor related to CMV disease (p < 0.01). The treatment with ganciclovir was successful in 17 of 19 symptomatic episodes. CONCLUSIONS: Active and symptomatic CMV infection in recipients of liver transplantation were very frequent in our study. The transplant from seropositive to seronegative and the use of anti-OKT3 antibodies increased the incidence of CMV disease. Ganciclovir appeared as a safe and, apparently, highly efficient drug.

[Insulin transference in 198 patients from 6 Latin American countries]. / Transferencia de insulinas en 198 pacientes de seis países latinoamericanos.

A multicentric, comparative, single-blind, randomized, prospective study was designed to evaluate the efficacy and safety of the transference from animal source insulins to rDNA human insulin in Latinamerican insulin-requiring diabetic patients. All the patients were on animal insulin for at least two months before inclusion. The patients were evaluated at the beginning, and at two and six weeks after inclusion. A total 198 patients completed the study and were considered evaluable: 94 were assigned to the animal insulin group, and 104 to the human insulin group. There were no statistically significant baseline differences between groups. The only statistically significant difference, detected at the end of the study, was a reduction in fasting blood glucose level in the human insulin group (animal insulin 212 +/- 95.3 initial vs 193 +/- 78.3 mg/dL final, p = 0.18; human insulin 198 +/- 86.8 vs 169 +/- 71.7, p = 0.025). There were no statistically significant initial-final changes in the rest of the parameters evaluated although a trend of reduction in glycohemoglobin levels was observed in both groups. There were more episodes of mild hipoglycemia in the human insulin group, and only one episode of severe unwarned hypoglycemia in the same group. We conclude that the transference of insulins in Latinamerican diabetic patients is effective and reasonably safe (with a dose adjustment when the change is made).

Panton-Valentine leukocidin-positive Staphylococcus aureus skin and soft tissue infections among children in an emergency department in Madrid, Spain.

Fifty-three children who attended the emergency department with community-associated (CA) Staphylococcus aureus skin and soft tissue infections (SSTIs) were enrolled in the study. Seven cases of infection (13.2%) were due to methicillin-resistant S. aureus (MRSA). Twelve of 46 available isolates (26.1%) were Panton-Valentine leukocidin (PVL)-positive. PVL-positive S. aureus SSTIs were more frequently associated with abscesses and cellulitis (75% vs. 38%, p 0.028), and more commonly required incision and drainage (75% vs. 21%, p 0.001). Most PVL-positive CA-MRSA isolates belonged to a single multilocus sequence type (ST8). In contrast, PVL-positive methicillin-susceptible S. aureus isolates belonged to four different sequence types (ST8, ST30, ST80, ST120).

[Evaluation of the effect of the dilution of urine on the toxicity and isolation of cytomegalovirus using the rapid shell-vial centrifugation culture technique]. / Evalución del efecto de la dilución de la orina sobre la toxicidad y el aislamiento de citomegalovirus con la técnica rápida de cultivo-centrifugación en viales.

Working with highly passaged MRC-5 fibroblast monolayers in a shell-vial culture-centrifugation assay for rapid detection of cytomegalovirus (CMV), a direct toxic effect of the urine on the monolayer was observed in as many as 44% of cases when vials were inoculated with undiluted specimens. This toxicity was reduced to only 6% by inoculation of shell-vials with samples diluted 1:1 with viral transport medium. The recovery of CMV in shell-vials was not significantly affected by the dilution factor, since five cases detected in conventional culture in tubes and not in vials were compensated by seven cases detected in vials and not in tubes.

High-level resistance to trimethoprim in Shigella sonnei associated with plasmid-encoded dihydrofolate reductase type I.

By DNA hybridization, the gene encoding dihydrofolate reductase type I was found in 58 of 59 highly trimethoprim-resistant clinical isolates of Shigella sonnei obtained from 1981 through 1987 in Madrid, Spain. No strain harbored the type II gene. In selected strains, the type I gene was demonstrated to be in a plasmid.

In vitro susceptibility of Brucella melitensis to new cephalosporins crossing the blood-brain barrier.

The in vitro susceptibilities of 83 clinical isolates of Brucella melitensis to seven cephalosporins and a monobactam were determined. Ceftizoxime, ceftriaxone, and cefotaxime were the most effective agents tested, with MICs ranging from 0.25 to 2 micrograms/ml. Moxalactam, cefoperazone, cefuroxime, and ceftazidime showed MICs between 4 and 64 micrograms/ml, with moxalactam being the most active agent in this group. Aztreonam showed poor activity, with MICs higher than 64 micrograms/ml.

[Meningitis caused by enterovirus in a pediatric hospital: experience in 1996]. / Meningitis por enterovirus en un hospital pediátrico: experiencia en 1996.

BACKGROUND: To know the incidence, etiology to the serotype level and epidemiological characteristics of cases of enteroviral meningitis diagnosed in a pediatric emergency service in 1996. METHODS: Inoculation of conventional cell cultures with samples of cerebrospinal fluid (CSF), throat swabs and stools taken from children with clinically suspected meningitis. RESULTS: Enterovirus was cultured from 17 of 65 (26.2%) CSF samples with pleocytosis (more than 10 cells/mm3) and from 12 of 268 (4.5%) biologically normal samples. The identified isolates of CSF (20 of 29) were: ECHO 30 (9), ECHO 11 (7), ECHO 9 (3) and ECHO 7 (1). Most (79%) isolates were obtained between March and June. CONCLUSIONS: Enteroviral meningitis was apparently more common in spring, although the number of samples was significantly lower during the summer months. The clinical cases were sporadic, due to at least four echoviruses serotypes. The isolation of enterovirus from CSF without pleocytosis is not exceptional (41% of our isolates).

Rapid diagnosis of group A streptococcal antigen extracted directly from swabs by an enzymatic procedure and used to detect pharyngitis.

Coagglutination after enzymatic digestion is a widely used, rapid procedure for serogrouping isolated colonies of beta-hemolytic streptococci. We tried to determine whether the same procedure could be used for the detection of group A streptococcal antigen directly from swabs used to take throat samples. This was achieved by incubating the swabs immersed in a small quantity of lytic extract obtained from cultures of the Maxted strain of Streptomyces griseus and testing the supernatant fluid by coagglutination. Of 538 throat swabs tested, blindly comparing the results of conventional cultures and rapid antigen detection, both tests were negative in 480 and both were positive in 49 swabs. In six cases, culture was positive and the rapid test was negative, but only one swab was from a patient with acute pharyngitis. In three cases, cultures were negative but the rapid test showed a strongly positive reaction. No special instructions were given to the physicians taking the samples. We conclude that this rapid antigen detection test, giving results in approximately 1 h, is an economic and reliable procedure for the detection of group A streptococcal antigen directly from throat samples.

Microtiter-adapted method that facilitates the Coombs test for brucellosis.

A simple method for carrying out the Coombs test with U-shaped microtiter plates instead of tubes is described. This technique provides reliable and more rapid testing of large numbers of sera in suspected cases of human brucellosis.

[Cytomegaloviruses in the bronchoalveolar lavage fluids of immunocompromised patients: microbiological results and clinical significance]. / Citomegalovirus en lavados broncoalveolares de pacientes inmunocomprometidos: resultados microbiológicos y significación clínica.

We have evaluated the microbiologic output and clinical significance of the detection of cytomegalovirus in 111 bronchoalveolar lavage specimens from immunosuppressed patients with pneumonitis. The samples were simultaneously processed by conventional tube culture and the rapid shell-vial centrifugation culture assay. Cytomegalovirus was recovered from 30 specimens (27%). The rapid shell-vial procedure was more sensitive than the tube culture, but in two cases cytomegalovirus was isolated only in tube cultures. Cytomegalovirus was considered clinically significant in only 3 from 13 HIV positive patients. All culture positive, HIV negative patients received treatment with ganciclovir. However, ganciclovir was never used on culture negative, HIV negative patients and cytomegalovirus related morbi-mortality was not found in these patients. A prospective study is needed to conclude if a cytomegalovirus negative culture also has a treatment exclusion value in HIV positive patients.

Efficacy and safety of imipenem/cilastatin in the empirical treatment of septicemia.

The clinical efficacy and safety of imipenem/cilastatin in the empirical treatment of adult non-immunocompromised patients with severe bacterial septicemia was studied in a prospective and open trial. The dosage of imipenem/cilastatin was 500 mg q 6 h. Of 58 patients included, 41 were evaluable for efficacy. In those patients, 35 had chronic underlying diseases and the foci of bacteremia were identified in 37; the most common ones being cardiovascular, urologic or intraabdominal infections. All isolated organisms were sensitive to imipenem with an MIC for 90% of the strains of 1 mg/l. Imipenem/cilastatin treatment resulted in rapid control of the infections in 39 of the 41 evaluable patients (95.5%). In the remaining two patients treatment had to be prematurely discontinued due to adverse effects. The causative bacterial strains were eradicated from blood in all patients who received more than one day of imipenem/cilastatin treatment but persisted sensitive to imipenem in peripheral foci in five patients (17%). Clinical and laboratory adverse reactions were noted in seven patients. In conclusion, imipenem/cilastatin was a well tolerated and effective empirical drug for treatment of septicemia.

A-549 is a suitable cell line for primary isolation of coxsackie B viruses.

A common receptor for coxsackie B virus and adenovirus has been described recently in cells of human and murine origin. Since the established cell line A-549 is suitable for adenoviruses, the potential use of A-549 cells for the isolation of coxsackie B viruses from clinical samples was investigated. All throat swabs sent to the laboratory between April 1998 and June 1999 were inoculated onto monolayers of MRC-5 and A-549 cells in tubes, and the enterovirus isolates obtained were typed. From April to June 1999, A-549 cells were compared prospectively to Buffalo green monkey (BGM) cells, considered as the most susceptible cell line for isolating coxsackie B viruses. Fifty-six out of 171 enterovirus isolates (33%) displayed a cytopathic effect (CPE) in the A-549 monolayer only, 48 isolates (28%) in the MRC-5 monolayer only, and 67 isolates (39%) in both cell lines. Most isolates that showed CPE in A-549 cells only (48 out of 56, 86%) were coxsackie B viruses, belonging to four different serotypes (B1, B2, B4, and B6). When BGM and A-549 cells were inoculated in parallel, both recovered the same number of coxsackie B isolates (n = 20), and the CPE was noted on approximately the same day. In conclusion, growth in A-549 but not MRC-5 cells identified coxsackie B viruses in most cases. A-549 was comparable to BGM for primary isolation of coxsackie B viruses.

[Yield of detection of Clostridium difficile toxin versus stool culture in the study of nosocomial diarrhea]. / Rentabilidad de la detección de toxina de Clostridium difficile frente a coprocultivo para el estudio de la diarrea nosocomial.

BACKGROUND: The aim of the study was to determine whether the detection of Clostridium difficile toxin in stools may be more profitable than conventional stool cultures for the etiologic study of nosocomial diarrhea and to analyze what risk factors favor the development of nosocomial diarrhea by C. difficile. METHODS: The presence of enteropathogens and A and B toxins of C. difficile were investigated (by monoclonal antibody enzymoimmunoassay) in stools of patients with nosocomial diarrhea. A series of patients simultaneously admitted without diarrhea were selected as the control group. RESULTS: During a 6 month period 92 patients with nosocomial diarrhea and 82 controls without diarrhea were studied. The C. difficile toxin was detected in 8 of these 174 patients (4.6%). Eight point seven percent of the nosocomial diarrheas were related with C. difficile while only 1% were due to an enteropathogen (Salmonella enteritidis). C. difficile toxin was not detected in any patient who did not have diarrhea. In comparison with the patients with diarrhea due to other causes, the patients with diarrhea by C. difficile had more frequently received antibiotics over the previous 7 days (57 vs 88%) and had been hospitalized for a longer time (> or = 7 days) (58 vs 88%) (p < 0.05). CONCLUSIONS: In the author's institution infection by Clostridium difficile is the most frequent cause of nosocomial infectious diarrhea, especially in patients admitted for a prolonged time or who receive antibiotics. The routine investigation of enteropathogens in the cases of nosocomial diarrhea does not seem justified while the detection of the A and B toxins of C. difficile may be more profitable.

Ganciclovir prophylaxis decreases frequency and severity of cytomegalovirus disease in seropositive liver transplant recipients treated with OKT3 monoclonal antibodies.

The efficacy of ganciclovir, given prophylactically, to prevent cytomegalovirus-related disease was evaluated in liver transplant recipients, mostly seropositive, under treatment with OKT3 monoclonal antibodies. The incidence of cytomegalovirus disease and visceral involvement was reduced, respectively, from 52 and 36% in the control group to 12 and 8% in the ganciclovir-treated patients. Leukopenia was a frequent (32%) side effect of ganciclovir administration.