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Respiratory syncytial virus (RSV) causes respiratory illness in children, immunosuppressed individuals and the elderly. However, the viral factors influencing the clinical outcome of RSV infections remain poorly defined. Defective viral genomes (DVGs) can suppress virus replication by competing for viral proteins and by stimulating antiviral immunity. We studied the association between detection of DVGs of the copy-back type and disease severity in three RSV A-confirmed cohorts. In hospitalized children, detection of DVGs in respiratory samples at or around the time of admission associated strongly with more severe disease, higher viral load and a stronger pro-inflammatory response. Interestingly, in experimentally infected adults, the presence of DVGs in respiratory secretions differentially associated with RSV disease severity depending on when DVGs were detected. Detection of DVGs early after infection associated with low viral loads and mild disease, whereas detection of DVGs late after infection, especially if DVGs were present for prolonged periods, associated with high viral loads and severe disease. Taken together, we demonstrate that the kinetics of DVG accumulation and duration could predict clinical outcome of RSV A infection in humans, and thus could be used as a prognostic tool to identify patients at risk of worse clinical disease.

Objectives Reactivation viremia is associated with adverse clinical outcomes and immune dysfunction in adults with sepsis. We determined the incidence of viremia and its association with clinical outcomes and immune paralysis phenotype in children with severe sepsis. Design Prospective cohort study. Setting Single academic PICU from September 2016 to March 2018. Patients Fifty-nine patients 2-17 years old treated for severe sepsis. Interventions None. Measurements and main results We performed real-time polymerase chain reaction assays on whole blood specimens to determine the incidence of cytomegalovirus. Cytomegalovirus was detected in three patients (5%). All patients with cytomegalovirus viremia were seropositive, with an incidence of 13% in this subset. We additionally performed Epstein-Barr virus and human herpesvirus-6 polymerase chain reaction assays on last available specimens and detected Epstein-Barr virus in 4% and human herpesvirus-6 in 30% of the study population. Overall, viremia was not associated with clinical outcomes or immune function in univariable analyses. However, viremia was associated with lower odds of complicated course (defined as death within 28 d or ≥ 2 organ dysfunctions at 7 d) after controlling for age, Pediatric Risk of Mortality III score, and blood transfusion (adjusted odds ratio, 0.08; 95% CI, 0.01-0.84; p = 0.04). Conclusions Children with severe sepsis had low rates of detectable viremia, which limited analyses of its association with clinical outcomes or immune paralysis phenotype. Given the rare occurrence of cytomegalovirus viremia, in particular, our study does not support a role for viremia as a biomarker of illness severity or as a modifiable risk factor of clinical outcomes for most patients. Future studies on the role of viremia in pediatric sepsis will need to consider the challenges posed by low rates of viremia in this population.

Two years after the COVID-19 pandemic started, the world continues to adapt to the profound effects that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has had on our lives. As the global crisis took hold, many looked to the medical technology/device industry for guidance and solutions. All while the industry itself, was disrupting its own processes and activities. In order to evolve and deliver accelerated innovation the industry had to be agile, resilient and collaborative with the broader healthcare community and technology partners. Now comes a time when we will start to see what changes were temporary and which ones will become part of the new process, but one thing is certain, we will not be going back to where we were pre-pandemic.

On behalf of the Clinical and Laboratory Standards Institute (CLSI), the Expert Panel on Microbiology would like to respond to the recent commentary by Kirby and colleagues voicing concerns related to verification of commercial antimicrobial susceptibility testing (AST) for new drugs that are introduced into the clinical laboratory (1).…

Background Geenius HIV 1/2 Supplemental Assay (Geenius; Bio-Rad Laboratories) is the only Food and Drug Administration-approved HIV-1/HIV-2 antibody differentiation test for the second step in the HIV laboratory testing algorithm. We characterized the occurrence of true HIV-1 and HIV-2 infections as well as false results in 6 US clinical laboratories using Geenius. Methods We examined routine HIV testing outcome data from the time the laboratories began using the algorithm with Geenius until September 30, 2017. We calculated the positive predictive value for Geenius HIV-1 and HIV-2 reactivity separately. Results Of 5,046,684 specimens tested, 41,791 had reactive antigen/antibody test results. Most specimens with reactive antigen/antibody results were HIV-1 antibody-positive established infections (n = 32,421), 1,865 of which also had indeterminate HIV-2 bands present. Ninety-three specimens were HIV-2 antibody positive or untypable for HIV-1/HIV-2 antibody. Acute HIV-1 infections were found in 528 specimens; 881 specimens lacked the nucleic acid test to determine the possibility of acute HIV-1 infection. False-positive antigen/antibody test results were present in 7505 specimens. Few specimens (n = 363) had false-positive antigen/antibody results with indeterminate Geenius and negative HIV-1 nucleic acid test results. The positive predictive values of Geenius reactivity were 99.4% for HIV-1 and 4.3% for HIV-2. Conclusions Routine testing using the laboratory testing algorithm with Geenius resulted in most specimens resolving as HIV negative or HIV-1 positive. The occurrence of indeterminate HIV-2 bands with a Geenius final assay interpretation of HIV-1 positive was more common than true HIV-2 infections. Reporting indeterminate HIV-2 results in this situation may cause confusion with interpreting HIV infection status.

The FecalSwab system (Copan Italia, Brescia, Italy) is a convenient alternative to bulk stool for the diagnosis of enteric pathogens. Although the U.S. Food and Drug Administration (FDA) approved for transport and culture of enteric bacterial pathogens, the FecalSwab has not been well assessed for its suitability with molecular platforms. In this study, we evaluated the FecalSwab as a specimen type for the BD Max system using the viral and bacterial enteric panels (BD Diagnostics, Baltimore, MD, USA)., The FecalSwab system (Copan Italia, Brescia, Italy) is a convenient alternative to bulk stool for the diagnosis of enteric pathogens. Although the U.S. Food and Drug Administration (FDA) approved for transport and culture of enteric bacterial pathogens, the FecalSwab has not been well assessed for its suitability with molecular platforms. In this study, we evaluated the FecalSwab as a specimen type for the BD Max system using the viral and bacterial enteric panels (BD Diagnostics, Baltimore, MD, USA). A total of 186 unpreserved stool specimens were collected and used to prepare matched bulk stool and FecalSwab samples. Performance was equivalent (P > 0.48) to bulk stool for all targets when 50 μl of FecalSwab specimen was loaded onto the BD Max assays. As stool specimens are often collected off-site from the clinical microbiology laboratory and require transport, we assessed the stability of stool specimens stored for up to 14 days at 4°C, 22°C, or 35°C to account for varying transportation conditions. Molecular detection for the majority of viral targets (excluding astrovirus) was unaffected (change in cycle threshold [ΔCT] ≤ 1) by sample storage temperature over the 2-week period; however, detection of enteric bacteria was variable if specimens were not refrigerated (22°C or 35°C). By demonstrating equivalent performance to matched bulk stool and maintaining molecular detection sensitivity when stored at 4°C, we suggest that the FecalSwab is a suitable specimen type for enteropathogen diagnostics on the BD Max system.

Purpose. Influenza A virus (FluA), influenza B virus (FluB) and respiratory syncytial virus (RSV) illnesses increase hospitalizations during seasonal epidemics. Methodology. To determine the utility of the Simplexa FluA/B & RSV Direct Assay (Direct Flu/RSV) and its impact on oseltamivir use, we offered this assay to emergency department (ED) patients with influenza-like illness. Results. Utilization of the Direct Flu/RSV provided a turnaround time (TAT) of 2 hours. Compared to the flu season prior to implementation of the Direct Flu/RSV, clinicians were more likely to prescribe 5 days of oseltamivir therapy for Direct Flu/RSV-positive patients in comparison to those with a negative test. Conclusions. Use of Direct Flu/RSV provides results rapidly, which leads to more appropriate use of oseltamivir. The ease of use of this assay and quick TAT allows for prompt decision-making, which is essential for patient care and effective disease control during the influenza season.

The diagnosis of pleural tuberculosis requires an invasive and time-consuming reference method. Polymerase chain reaction (PCR) is rapid, but validation in pleural tuberculosis is still weak.To establish the operating characteristics of real-time polymerase chain reaction (RT-PCR) hybridization probes for the diagnosis of pleural tuberculosis.The validity of the RT-PCR hybridization probes was evaluated compared to a composite reference method by a cross-sectional study at the Hospital Universitario de la Samaritana. 40 adults with lymphocytic pleural effusion were included. Pleural tuberculosis was confirmed (in 9 patients) if the patient had at least one of three tests using the positive reference method: Ziehl-Neelsen orThe RT-PCR hybridization probes showed a sensitivity of 66.7% (95% CI: 33.2%-90.7%) and a specificity of 93.5% (95% CI: 80.3%-98.9%). The PPV was 75.0% (95% CI: 38.8%-95.6%) and a NPV of 90.6% (95% CI: 76.6%-97.6%). Two false positives were found for the test, one with pleural mesothelioma and the other with chronic pleuritis with mesothelial hyperplasia.The RT-PCR hybridization probes had good specificity and acceptable sensitivity, but a negative value cannot rule out pleural tuberculosis.El diagnóstico de tuberculosis pleural requiere un método de referencia invasivo y demorado. La reacción en cadena de la polimerasa es rápida, pero su validación en tuberculosis pleural aún es débil.Establecer las características operativas de la reacción en cadena de la polimerasa en tiempo real (RT-PCR) sondas de hibridación para el diagnóstico de tuberculosis pleural.Se evaluó la validez de la RT-PCR sondas de hibridación comparada con un método de referencia compuesto mediante un estudio transversal en el Hospital Universitario de la Samaritana. Se incluyeron 40 adultos con derrame pleural linfocitario. Tuberculosis pleural fue confirmada (en 9 pacientes) si el paciente tenía mínimo una de tres pruebas del método de referencia positiva: Ziehl-Neelsen o cultivo paraLa RT-PCR sondas de hibridación mostró una sensibilidad del 66.7% (IC 95%: 33.2%-90.7%) y una especificidad del 93.5% (IC 95%: 80.3%-98.9%). El VPP fue de 75.0% (IC 95%: 38.8%-95.6%) y un VPN de 90.6% (IC 95%: 76.6%-97.6%). Se encontraron dos falsos positivos para la prueba, uno con mesotelioma pleural y otro con pleuritis crónica con hiperplasia mesotelial.La RT-PCR sondas de hibridación tuvo una buena especificidad y una aceptable sensibilidad, pero un valor negativo no puede descartar tuberculosis pleural.

Background There are currently no FDA-cleared assays with a dual-claim for both diagnosis and monitoring of HIV-1. The Aptima HIV-1 Quant Dx Assay on the Panther platform (Panther) is the first commercially available test that is CE-marked for both HIV-1 diagnosis and monitoring, but only FDA-cleared for HIV-1 monitoring. Objective To evaluate the Panther assay for use as a qualitative and quantitative HIV-1 assay in a pediatric population, including patients younger than 24 months old, and review its effect on laboratory efficiency and hands-on-time following its implementation. Study Design 100 patient specimens previously tested on the Abbott m2000 RealTime HIV-1 assay (RealTime) and 185 patient specimens previously tested on the Aptima HIV-1 RNA Qualitative Assay (RNA Qual) were tested on the Panther. Verification panels were used to establish precision and linearity. In addition, 268 samples from 134 patients under 24 months of age were also evaluated on the Panther. Results Overall agreement between the Panther and RealTime assays was 83 %. The mean difference between the two methods was 0.10 Log copies/mL. All Panther measurements were linear across the dynamic range (R2 = 0.999). The Panther and RNA Qual assays showed 100 % agreement. Implementation of the assay opened 600 sq. ft. of space, saved 0.4 FTE and reduced hands-on-time by 70 %. Conclusions The Panther assay is an excellent option for HIV-1 qualitative detection and quantitative testing in pediatric patients, including those under 24 months of age. HIV testing on one platform has opened up space in the clinical laboratory and reduced hands on testing time.

Information about human adenovirus (HAdV) infection in solid organ transplant (SOT) recipients is limited. We aimed to describe HAdV infection epidemiology and outcomes in a single-center retrospective cohort during the era of polymerase chain reaction (PCR) availability. SOT recipients transplanted at the Children’s Hospital of Philadelphia 2004—2013 were followed for 180 days post-transplant. HAdV infection was defined as a positive HAdV PCR from a clinical specimen. HAdV disease was defined by organ-specific radiologic and/or laboratory abnormalities. No HAdV surveillance protocols were employed during the study period; testing was solely per clinician discretion. Progression of HAdV infection was defined as HAdV disease or ≥1-log viral load increase since a corresponding site’s first positive specimen. Of the assembled 425 SOT recipients, 227 (52.6%) had ≥1 HAdV PCR. Twenty-four (10.6%) had ≥1 HAdV-positive PCR. HAdV-positive subjects were younger than uninfected subjects (2.0 years vs. 6.5, p=0.001). Infection incidence rates were highest in liver recipients (15.3%), followed by heart (8.6%), kidney (8.3%), and lung (4.2%). Four subjects (16.7%) met HAdV disease criteria at virus detection. Five subjects (20.8%) had progression of HAdV infection. All-cause mortality rates in positive and negative subjects were 0% and 3.9%, respectively. HAdV infection was infrequently detected in SOT recipients. Over one-third of HAdV-positive patients met disease criteria at detection or had infection progression, but none died. This low all-cause mortality raises questions about benefits of HAdV surveillance. Larger multi-center studies are needed to assess incidence variance by center and comparative effectiveness of therapeutic interventions.

Chlamydia trachomatis and Neisseria gonorrhoeae are the two most common causes of sexually transmitted disease in the United States. Studies in adults, mostly in men who have sex with men, have shown that the prevalence of C. trachomatis and N. gonorrhoeae infections is much higher in extragenital sources compared to urogenital sources. A similar large sample of data on the burden of C. trachomatis and N. gonorrhoeae infections by anatomic site is lacking in children. We retrospectively analyzed data from 655 patients tested for C. trachomatis (887 specimens) and N. gonorrhoeae (890 specimens) at the Children’s Hospital of Philadelphia. We restricted the analysis to include patients between 2 and 17 years of age that had all three sources (urine, oropharynx, and rectum) collected at the same visit. The final data set included specimens from all three sources from 148 and 154 patients for C. trachomatis and N. gonorrhoeae, respectively. Specimens were tested for C. trachomatis and N. gonorrhoeae using a Gen-Probe Aptima Combo 2 assay. The burden of C. trachomatis and N. gonorrhoeae infection was significantly higher in the 14- to 17-year age group (24.7%, P = 0.041; 25.8%; P = 0.001) compared to the 10- to 13-year (5.9%; 5.6%), 6- to 9-year (4.6%; 4.6%), and 2- to 5-year (8.3%; 0%) age groups, respectively. The positivity rate for C. trachomatis was highest for rectal (16.2%), followed by urine (5.4%) and oropharyngeal (0.7%) sites. The positivity rate for N. gonorrhoeae was highest for rectal sites (10.4%), followed by oropharyngeal (9.7%) and urine (1.9%) sites. The source with highest diagnostic yield is rectum for C. trachomatis and rectum and oropharynx for N. gonorrhoeae. Hence, extragenital screening is critical for the comprehensive detection of C. trachomatis and N. gonorrhoeae in the pediatric population.

Introduction Enterovirus may result in a devastating chronic encephalitis in immunocompromised patients, particularly in patients with X-linked agammaglobulinemia. Prognosis for patients with chronic enterovirus encephalitis is poor, almost invariably resulting in mortality without specific treatment. There are currently no approved antiviral agents for enterovirus, but the antidepressant drug fluoxetine has been identified through library-based compound screening as a potential anti-enteroviral agent in vitro . However, use of fluoxetine has not previously been studied in humans with enteroviral disease. Patient Description A five year old boy with X-linked agammaglobulinemia presented with progressive neurological deterioration and was found to have chronic enterovirus encephalitis by brain biopsy. He failed to respond to standard treatment with high dose intravenous immunoglobulin, but showed stabilization and improvement following treatment with fluoxetine. Conclusions This is the first report to describe the use of fluoxetine as a potential therapy for chronic enterovirus infection. Further investigation of fluoxetine as a treatment option for chronic enterovirus encephalitis is necessary.

• HAdV infection should be considered as a source of disease in the pediatric solid organ transplant population, causing severe disseminated disease or having an atypical presentation including hepatic focal lesions with absence of liver necrosis.

Intravenous immunoglobulin (IVIg) therapy is increasingly used in the pediatric population, in particular among children with immune-compromising conditions. Pooled immunoglobulin products are routinely tested for hepatitis B surface antigen (HBsAg) and nucleic acid; however, screening for hepatitis B core antibody (anti-HBc) is not commonly performed. Thus, the administration of IVIg containing anti-HBc to children with immune-compromising conditions may complicate the interpretation of hepatitis B serologic testing in that a positive anti-HBc test may represent passive transfer of antibody from IVIg or may indicate resolved or chronic hepatitis B infection. Due to the risk of hepatitis B reactivation in immunocompromised patients, a positive anti-HBc test must be carefully considered. As part of a quality improvement initiative, we identified and reviewed the records of all pediatric patients at our institution who tested positive for anti-HBc over an 18-month period. Of 44 total patients with positive anti-HBc tests, we found that 22 (50%) had previously received IVIg in the preceding 4 months. All but one of these, 21/22 (95%), went on to receive immunosuppressive therapy (IS). Among the patients who received IS, 19 (86%) had not undergone hepatitis B serologic testing prior to IVIg administration and 16 (73%) did not have subsequent testing to distinguish between passive acquisition of anti-HBc from IVIg and chronic hepatitis B infection. Our single-center experience reveals that a high proportion of positive anti-HBc tests in children are presumed to be because of the passive antibody transfer from IVIg. However, a low proportion of patients undergo confirmatory testing, despite the risk of hepatitis B reactivation during IS. We thus propose a risk-based algorithm for interpretation and monitoring of hepatitis B testing in immunocompromised children.

BACKGROUND Human adenoviruses (HAdVs) are associated with significant morbidity and death after hematopoietic cell transplantation (HCT). In this study, we sought to determine the incidence of HAdV infection among pediatric HCT recipients in the polymerase chain reaction (PCR) testing era, identify risk factors for viremia among patients undergoing HAdV surveillance, and assess the effectiveness of preemptive cidofovir. METHODS A single-center retrospective cohort of patients who underwent a transplant within a 10-year period was assembled. The incidence of and outcomes of patients with HAdV infection and disease were determined by PCR results and chart review. A Cox regression model was used for surveilled allogeneic HCT recipients to identify factors associated with viremia. We also used a discrete-time failure model with inverse probability treatment weights to assess the effectiveness of preemptive cidofovir for infection. RESULTS Among 572 HCT recipients, 76 (13.3%) had ≥1 sample that was HAdV PCR positive (3.5% of autologous HCT recipients and 19.7% of allogeneic HCT recipients). Among 191 allogeneic HCT recipients under surveillance, 58 (30.4%) had HAdV detected from any source, and 50 (26.2%) specifically had viremia. The mortality rate was higher in allogeneic HCT recipients with HAdV infection versus those without infection (25.9% vs 11.3%; P = .01). Factors associated with infection included an age of 6 to 12 years, an absolute lymphocyte count of

We compared an FDA cleared molecular meningitis/encephalitis panel to lab developed viral PCRs and bacterial culture. Of the 67 viral PCR or bacterial culture-positive samples, 92.5% were positive for the same target by the panel. Of the 66 negative samples tested, no targets were detected by the panel, for an agreement of 96.2%.

Background Hospitals use antibiograms to guide optimal empiric antibiotic therapy, reduce inappropriate antibiotic usage, and identify areas requiring intervention by antimicrobial stewardship programs. Creating a hospital antibiogram is a time-consuming manual process that is typically performed annually. Objective We aimed to apply visual analytics software to electronic health record (EHR) data to build an automated, electronic antibiogram (“e-antibiogram”) that adheres to national guidelines and contains filters for patient characteristics, thereby providing access to detailed, clinically relevant, and up-to-date antibiotic susceptibility data. Methods We used visual analytics software to develop a secure, EHR-linked, condition- and patient-specific e-antibiogram that supplies susceptibility maps for organisms and antibiotics in a comprehensive report that is updated on a monthly basis. Antimicrobial susceptibility data were grouped into nine clinical scenarios according to the specimen source, hospital unit, and infection type. We implemented the e-antibiogram within the EHR system at Children's Hospital of Philadelphia, a tertiary pediatric hospital and analyzed e-antibiogram access sessions from March 2016 to March 2017. Results The e-antibiogram was implemented in the EHR with over 6,000 inpatient, 4,500 outpatient, and 3,900 emergency department isolates. The e-antibiogram provides access to rolling 12-month pathogen and susceptibility data that is updated on a monthly basis. E-antibiogram access sessions increased from an average of 261 sessions per month during the first 3 months of the study to 345 sessions per month during the final 3 months. Conclusion An e-antibiogram that was built and is updated using EHR data and adheres to national guidelines is a feasible replacement for an annual, static, manually compiled antibiogram. Future research will examine the impact of the e-antibiogram on antibiotic prescribing patterns.

Mycobacterium llatzerense was cultured from a subdiaphragmatic abscess. To our knowledge, this is the first report of isolation of this rapidly growing mycobacterium from a human. Growth characteristics and antimicrobial susceptibilities different from those previously reported for environmental isolates were observed. Copyright © 2014, American Society for Microbiology. All Rights Reserved., M.G. and J.L. thank the Spanish MINECO for financial support through projects CGL2011-24318 and Consolider CSD2009-00006. M.G. was supported by a postdoctoral contract from the University of the Balearic Islands, with funds from Spanish Ministry of Education, Culture and Sports through the International Excellence Campus

We transitioned laboratory-developed PCR assays for herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) to the BD Max system by using BD Max open system reagents. After optimization, the agreement with the reference PCR assay was 100% (123/123) for HSV-1, 96.7% (119/123) for HSV-2, and 100% (60/60) for VZV using retrospective clinical samples.

Mixed-population (heterogeneous) enterococcal bacteremia (MEB) is rarely reported. Based on one occasion in which Vitek2 missed a vancomycin-resistant subpopulation isolated from a patient, we developed a simple method to detect this subpopulation and determined MEB frequency. The four patients presented here had either Enterococcus faecium or Enterococcus faecalis bacteremia caused by both vancomycin-resistant enterococci (VRE) and vancomycin-susceptible enterococci (VSE). No prior common antibiotic therapy was observed, and bacteremia resolved with daptomycin, gentamicin, and/or linezolid treatment. In two cases, VRE presence was missed by Vitek2. To detect the VRE subpopulation, tryptic soy broth was inoculated from positive blood cultures and a saline suspension was inoculated to a vancomycin (6-μg/ml) (V6) plate. Two isolates from each patient were studied further. Relatedness was assessed by multilocus sequence typing, fitness was evaluated by growth curve and competition assays, and vanA presence was determined by PCR. MEB represented ∼5% of all enterococcal bacteremias. All VRE subpopulations grew on V6 plates but were missed in two instances by Vitek2. VRE and VSE isolates from each patient were closely related and did not differ in overall fitness. All four VRE isolates and 2/4 VSE isolates were vanA positive. MEBs occur regardless of prior antimicrobial therapy, are relatively common in our hospital, and are important to detect. As far as we know, this study is the first to report heterogeneous E. faecalis bacteremia. There is a simple method to detect VRE subpopulations that may be missed by Vitek2.

Background In the United States, a new HIV diagnostic algorithm has been proposed that uses an HIV-1/HIV-2 antibody differentiation immunoassay instead of Western blot or immunofluoresence for confirmatory testing. Objectives To evaluate the Multispot HIV-1/HIV-2 Rapid Test (Multispot) as an alternative to Western blot analysis for confirmation of HIV infection. Study design A series of 205 serum and plasma specimens positive for HIV-1 or HIV-2 were used to compare the performance of Multispot to a standard HIV-1 Western blot. Positive samples included 63 specimens from patients > 18 months of age, 33 proficiency survey specimens, and 109 specimens from nine commercial seroconversion and performance panels. In addition, 63 specimens from 51 HIV-exposed, uninfected children ≤ 18 months of age in various stages of seroreversion and 192 HIV-negative samples were tested. Specimens were initially screened using a 4th generation HIV Ag/Ab Combo assay. Results Multispot readily discriminated between individuals with HIV-1 or HIV-2 infection and those who were uninfected. Of the 205 samples repeatedly reactive by the 4th generation screening assay, infection status was correctly confirmed by Multispot in 83.9% (172/205) compared to 68.8% (141/205) for Western blot. Multispot detected HIV-1 earlier in 27.6% of low-titer antibody specimens called indeterminate by Western blot, and effectively reduced the number of indeterminate results in seroreverting HIV-1 exposed, uninfected infants and for HIV-2 infections misinterpreted as indeterminate or positive by HIV-1 Western blot. Conclusions Multispot offers speed and simplicity over Western blot and has an excellent performance for differentiation and confirmation of antibodies to HIV-1 and HIV-2.

Fluoroquinolones are broad spectrum antibiotics commonly used in the treatment of infections.Resistance profiles of coccus bacteria to fluoroquinolones were evaluated in isolates of Streptococcus pneumoniae, Staphylococcus aureus, coagulase negative staphylococci and Enterococcus spp. The samples were recovered from Colombian hospitals between 1994 and 2004.The minimal inhibitory concentration of ciprofloxacin, moxifloxacin and gatifloxacin was determined in 270 clinical isolates of S. pneumoniae, 348 of S. aureus, 176 of coagulase negative staphylococci and 123 of coagulase-negative enterococci. The minimal inhibitory concentration of levofloxacin was also determined in all isolates of methicillin-resistant S. aureus. An agar diffusion susceptibility test with disks of levofloxacin and ofloxacin was also applied to all isolates of S. pneumoniae.A total of 269 (99.6%) isolates of S. pneumoniae were susceptible to moxifloxacin and gatifloxacin. For levofloxacin and ofloxacin, resistance in S. pneumoniae was found in 1.5% and 8.9% of isolates, respectively. The ciprofloxacin minimal inhibitory concentration was4 microg/ml in 15.9% of pneumococcal isolates. The rates of resistance to ciprofloxacin, gatifloxacin and moxifloxacin in the 348 S. aureus isolates were 55.4%, 54.9% and 52.6%, respectively; increasing to 92.3%, 91.3% and 87.5%, respectively in methicillin resistant isolates. Resistance to levofloxacin was found in 91.8% of MRSA isolates. The rates of resistance to ciprofloxacin, gatifloxacin and moxifloxacin in coagulase negative staphylococci and vancomycin-susceptible enterococci were between 25.6% and 31.8%. All vancomycin-resistant enterococci were resistant to all fluroquinolones tested.The newer fluoroquinolones have maintained effective activity against clinical isolates of S. pneumoniae. The rates of fluoroquinolone resistance in S. aureus were very high, particularly in methicillin resistant isolates (approaching 100%).

In order to overcome some of the limitations of conventional microbiological techniques in the diagnosis of human brucellosis, a simple PCR-enzyme-linked immunosorbent assay (PCR-ELISA) was developed. After amplification of a 223-bp sequence of a gene that codes for the synthesis of an immunogenetic membrane protein specific for the Brucella genus (BCSP31), the digoxigenin-labeled amplified product was hybridized with a biotinylated capture probe which was complementary to the inner part of the amplicon. The hybrid was captured on streptavidin-coated microtiter plates and detected by using an antidigoxigenin Fab-peroxidase conjugate. The detection limit of the PCR-ELISA in a background of 3.5 μg of human genomic DNA was 10 fg (two bacterial cells). The PCR-ELISA showed an analytical sensitivity higher than that of ethidium bromide staining and equal to that obtained by conventional PCR followed by dot blot hybridization. In 59 peripheral blood samples from 57 consecutive patients with active brucellosis and 113 control samples, the PCR-ELISA was found to be 94.9% sensitive and 96.5% specific, whereas the sensitivity of the blood culture was only 70.1%. Since the assay can be performed in 1 day, is very reproducible, is easily standardized, and avoids the risk of infection in laboratory workers, this PCR-ELISA seems to be a practical and reliable tool for the diagnosis of human brucellosis.