Atypical Mutational Spectrum of SARS-CoV-2 Replicating in the Presence of Ribavirin.

We report that ribavirin exerts an inhibitory and mutagenic activity on SARS-CoV-2-infecting Vero cells, with a therapeutic index higher than 10. Deep sequencing analysis of the mutant spectrum of SARS-CoV-2 replicating in the absence or presence of ribavirin indicated an increase in the number of mutations, but not in deletions, and modification of diversity indices, expected from a mutagenic activity. Notably, the major mutation types enhanced by replication in the presence of ribavirin were A→G and U→C transitions, a pattern which is opposite to the dominance of G→A and C→U transitions previously described for most RNA viruses. Implications of the inhibitory activity of ribavirin, and the atypical mutational bias produced on SARS-CoV-2, for the search for synergistic anti-COVID-19 lethal mutagen combinations are discussed.

Antibody response in patients admitted to the hospital with suspected SARS-CoV-2 infection: results from a multicenter study across Spain.

AIM: To evaluate the serological response against SARS-CoV-2 in a multicenter study representative of the Spanish COVID pandemic. METHODS: IgG and IgM + IgA responses were measured on 1466 samples from 1236 Spanish COVID-19 patients admitted to the hospital, two commercial ELISA kits (Vircell SL, Spain) based on the detection of antibodies against the viral spike protein and nucleoprotein, were used. RESULTS: Approximately half of the patients presented antibodies (56.8% were IgM + IgA positive and 43.0% were IgG positive) as soon as 2 days after the first positive PCR result. Serological test positivity increased with time from the PCR test, and 10 days after the first PCR result, 91.5% and 88.0% of the patients presented IgM + IgA and IgG antibodies, respectively. CONCLUSION: The high values of sensitivity attained in the present study from a relatively early period of time after hospitalization support the use of the evaluated serological assays as supplementary diagnostic tests for the clinical management of COVID-19.

Synergistic lethal mutagenesis of hepatitis C virus.

Lethal mutagenesis is an antiviral approach that consists in extinguishing a virus by an excess of mutations acquired during replication in the presence of a mutagenic agent, often a nucleotide analogue. One of its advantages is its broad spectrum nature that renders the strategy potentially effective against emergent RNA viral infections. Here we describe synergistic lethal mutagenesis of hepatitis C virus (HCV) by a combination of favipiravir (T-705) and ribavirin. Synergy has been documented over a broad range of analogue concentrations using the Chou-Talalay method as implemented in the CompuSyn graphics, with average dose reduction index (DRI) above 1 (68.02±101.6 for favipiravir, and 5.83±6.07 for ribavirin), and average combination indices (CI) below 1 (0.52±0.28). Furthermore, analogue concentrations that individually did not extinguish high fitness HCV in ten serial infections, when used in combination they extinguished high fitness HCV in one to two passages. Although both analogues display a preference for G→A and C→U transitions, deep sequencing analysis of mutant spectra indicated a different preference of the two analogues for the mutation sites, thus unveiling a new possible synergy mechanism in lethal mutagenesis. Prospects of synergy among mutagenic nucleotides as a strategy to confront emerging viral infections are discussed.

Evaluation of the use of sonication of retrieved implants for the diagnosis of prosthetic joint infection in a routine setting.

In order to evaluate the usefulness of sonication of retrieved implants for the diagnosis of prosthetic joint infection (PJI) in a large group of patients in a routine setting, we designed a 3-year retrospective study. Patients were classified into two groups: those meeting the clinical criteria of PJI and those that did not (control group). Two hundred patients and 276 samples were included. The types of infection were early (n = 44), delayed (n = 53), positive intraoperative cultures (n = 13) and late-acute (n = 8). The culture sensitivities of sonicate fluid, periprosthetic tissue, synovial fluid and combination of periprosthetic tissue and/or synovial fluid were 69.5, 52.8, 54.8 and 60.2%, respectively. The specificities were 97.6, 90.3, 93.0 and 89.9%, respectively. Sonicate fluid culture of implants was more sensitive than peri-implant tissue, synovial fluid and combination of periprosthetic tissue and/or synovial fluid for all infection types, though it was especially useful in delayed infection: 91.3% vs. 60.0% (p = 0.0015), 63.2% (p = 0.0005) and 66.7% (p = 0.0001), respectively. When sonicate fluid culture of implants was performed in addition to conventional cultures, the sensitivity increased significantly in total (from 60.2 to 77.1%) and delayed PJI (from 45.1 to 71.7%). On the other hand, for early PJI, sonicate fluid culture of prosthesis was not superior to conventional diagnostic methods.

Evaluation of a commercial multiplex PCR (Unyvero i60®) designed for the diagnosis of bone and joint infections using prosthetic-joint sonication.

BACKGROUND: The development of sonication protocols over the last few years has improved the sensitivity of conventional cultures for the diagnosis of prosthetic-joint infection (PJI). However, the development of a new, specifically designed kit for the molecular diagnosis of PJI could provide a major improvement in this field. METHODS: Prostheses retrieved from patients who underwent implant removal from May 2014 to May 2015 were sent for culture, and processed according to a previously defined protocol that included sonication. Furthermore, 180 microlitres of sonication fluid were used to carry out the multiplex PCR test (Unyvero i60 system®). A comparison of the sensitivity, specificity, positive (PPV) and negative (NPV) predictive value, was performed. The study was approved by the Clinical Research Ethics Committee. RESULTS: The analysis included 88 prostheses from 68 patients (1.29 prostheses/patient). The type of prostheses studied were knee (n=55), total hip (n=26), partial hip (n=5), and shoulder (n=2). Twenty-nine patients were diagnosed with a PJI (15 delayed, 12 acute, and 2 haematogenous infections). In 24 cases, the result of the PCR was positive, all but 1 corresponding to patients with clinical criteria of PJI. Nine resistance mechanisms were detected from 5 samples. The Unyvero i60 system® showed slightly better results than traditional culture in terms of specificity and PPV. CONCLUSIONS: The Unyvero i60 system® may play a role in rapid diagnosis of PJI, due to its high specificity and PPV. However, despite these results, cultures have to be performed to detect organisms not detected by the system.

Anorectal Lymphogranuloma Venereum in Madrid: A Persistent Emerging Problem in Men Who Have Sex With Men.

BACKGROUND: Since 2003, outbreaks of lymphogranuloma venereum (LGV) with anorectal syndrome have been increasingly recognized in many Western countries. All of them have been classified as LGV serovar L2b, mainly occurring in human immunodeficiency virus (HIV)-infected men who have had sex with men (MSM). We describe a series of 26 diagnosed cases of LGV proctitis in downtown Madrid, Spain, in 2014, after implementing routine diagnostic procedures for this disease in symptomatic MSM. METHODS: We conducted an observational study of patients with symptomatic proctitis attending an outpatient infectious diseases clinic in Madrid, Spain during calendar year 2014. Clinical, epidemiological, laboratory, and therapeutic data were gathered and analyzed. RESULTS: Twenty-six patients were included in the analysis. All were MSM, and 24 of them were HIV-positive. All patients reported having acute proctitis symptoms including tenesmus (85%), pain (88%), constipation (62%), or anal discharge (96%). Proctoscopy showed mucopurulent exudate (25 patients [96%]), and rectal bleeding, with mucosal erythema and/or oedema in all cases. Rectal swabs were obtained from all patients, and LGV serovar L2 was confirmed in all of them. The cure rate was 100% after standard treatments with doxycycline 100 mg twice per day for 3 weeks. Simultaneous rectal infections with other sexually transmitted pathogens (gonorrhoea, herpes simplex virus, Mycoplasma genitalium) and systemic sexually transmitted diseases (STDs) (syphilis, acute HIV, and hepatitis C infections) were also documented in 12 patients (46%), but these co-infections did not appear to influence the clinical manifestations of LGV. CONCLUSIONS: Anorectal LGV is a common cause of acute proctitis and proctocolitis among HIV-infected MSM who practice unprotected anal sex, and it is frequently associated with other rectal STDs. The implementation of routine screening and prompt diagnosis of these rectal infections should be mandatory in all clinical settings attended by HIV and STD patients.

SARS-CoV-2 mutant spectra as variant of concern nurseries: endless variation?

Introduction: SARS-CoV-2 isolates of a given clade may contain low frequency genomes that encode amino acids or deletions which are typical of a different clade. Methods: Here we use high resolution ultra-deep sequencing to analyze SARS-CoV-2 mutant spectra. Results: In 6 out of 11 SARS-CoV-2 isolates from COVID-19 patients, the mutant spectrum of the spike (S)-coding region included two or more amino acids or deletions, that correspond to discordant viral clades. A similar observation is reported for laboratory populations of SARS-CoV-2 USA-WA1/2020, following a cell culture infection in the presence of remdesivir, ribavirin or their combinations. Moreover, some of the clade-discordant genome residues are found in the same haplotype within an amplicon. Discussion: We evaluate possible interpretations of these findings, and reviewed precedents for rapid selection of genomes with multiple mutations in RNA viruses. These considerations suggest that intra-host evolution may be sufficient to generate minority sequences which are closely related to sequences typical of other clades. The results provide a model for the origin of variants of concern during epidemic spread─in particular Omicron lineages─that does not require prolonged infection, involvement of immunocompromised individuals, or participation of intermediate, non-human hosts.

Synergism between remdesivir and ribavirin leads to SARS-CoV-2 extinction in cell culture.

BACKGROUND AND PURPOSE: There is a need for effective anti-COVID-19 treatments, mainly for individuals at risk of severe disease such as the elderly and the immunosuppressed. Drug repositioning has proved effective in identifying drugs that can find a new application for the control of coronavirus disease, in particular COVID-19. The purpose of the present study was to find synergistic antiviral combinations for COVID-19 based on lethal mutagenesis. EXPERIMENTAL APPROACH: The effect of combinations of remdesivir and ribavirin on the infectivity of SARS-CoV-2 in cell culture has been tested. Viral populations were monitored by ultra-deep sequencing, and the decrease of infectivity as a result of the treatment was measured. KEY RESULTS: Remdesivir and ribavirin exerted a synergistic inhibitory activity against SARS-CoV-2, quantified both by CompuSyn (Chou-Talalay method) and Synergy Finder (ZIP-score model). In serial passage experiments, virus extinction was readily achieved with remdesivir-ribavirin combinations at concentrations well below their cytotoxic 50 value, but not with the drugs used individually. Deep sequencing of treated viral populations showed that remdesivir, ribavirin, and their combinations evoked significant increases of the number of viral mutations and haplotypes, as well as modification of diversity indices that characterize viral quasi-species. CONCLUSION AND IMPLICATIONS: SARS-CoV-2 extinction can be achieved by synergistic combination treatments based on lethal mutagenesis. In addition, the results offer prospects of triple drug treatments for effective SARS-CoV-2 suppression.

Kingella kingae keratitis.

We present the first two cases of Kingella kingae keratitis in adults. This species is a rare isolate from ophthalmic samples for which final identification was obtained with matrix-assisted laser desorption ionization-time of flight mass spectrometry. One of the patients recovered uneventfully with topical therapy. Results from the second patient cannot be confirmed as he was lost to follow-up.

[Enteric fever in Madrid. A review of the last 30 years]. / Fiebre entérica en Madrid. Una revisión de los últimos 30 años.

BACKGROUND: Our aim is analyze the epidemiological factors of enteric fever in Madrid (Spain) over the last 30 years. MATERIAL AND METHODS: A retrospective review was conducted on cases of typhoid and paratyphoid fever studied in the Fundación Jiménez Díaz (Madrid) between 1980 and 2010. Two similar periods in time were studied (P1: 1980-1993; P2: 1994-2010). RESULTS: There was a total of 61 confirmed cases of enteric fever: 51 (84%) were typhoid and 10 were paratyphoid: 45 patients were native Spanish (40 belonging to P1) and 16 were immigrants (15 in P2, with 11 of them coming from the Indian sub-continent). CONCLUSION: Enteric fever must be present in the differential diagnosis of persistent fever without clear focus in immigrants, mainly from the Indian subcontinent, and travelers from endemic areas.

PCR-hybridization after sonication improves diagnosis of implant-related infection.

PURPOSE: We wanted to improve the diagnosis of implant-related infection using molecular biological techniques after sonication. METHODS: We studied 258 retrieved implant components (185 prosthetic implants and 73 osteosynthesis implants) from 126 patients. 47 patients had a clinical diagnosis of infection (108 components) and 79 patients did not (150 components). The fluids from sonication of retrieved implants were tested in culture and were also analyzed using a modified commercial PCR kit for detection of Gram-positive and Gram-negative bacteria (GenoType BC; Hain Lifescience) after extraction of the DNA. RESULTS: 38 of 47 patients with a clinical diagnosis of infection were also diagnosed as being infected using culture and/or PCR (35 by culture alone). Also, 24 patients of the 79 cases with no clinical diagnosis of infection were identified microbiologically as being infected (4 by culture, 16 by PCR, and 4 by both culture and PCR). Comparing culture and PCR, positive culture results were obtained in 28 of the 79 patients and positive PCR results were obtained in 35. There were 21 discordant results in patients who were originally clinically diagnosed as being infected and 28 discordant results in patients who had no clinical diagnosis of infection. INTERPRETATION: For prosthetic joint infections and relative to culture, molecular detection can increase (by one tenth) the number of patients diagnosed as having an infection. Positive results from patients who have no clinical diagnosis of infection must be interpreted carefully.

Vaccine breakthrough infections with SARS-CoV-2 Alpha mirror mutations in Delta Plus, Iota, and Omicron.

Replication of SARS-CoV-2 in the human population is defined by distributions of mutants that are present at different frequencies within the infected host and can be detected by ultra-deep sequencing techniques. In this study, we examined the SARS-CoV-2 mutant spectra of amplicons from the spike-coding (S-coding) region of 5 nasopharyngeal isolates derived from patients with vaccine breakthrough. Interestingly, all patients became infected with the Alpha variant, but amino acid substitutions that correspond to the Delta Plus, Iota, and Omicron variants were present in the mutant spectra of the resident virus. Deep sequencing analysis of SARS-CoV-2 from patients with vaccine breakthrough revealed a rich reservoir of mutant types and may also identify tolerated substitutions that can be represented in epidemiologically dominant variants.

SARS-CoV-2 Mutant Spectra at Different Depth Levels Reveal an Overwhelming Abundance of Low Frequency Mutations.

Populations of RNA viruses are composed of complex and dynamic mixtures of variant genomes that are termed mutant spectra or mutant clouds. This applies also to SARS-CoV-2, and mutations that are detected at low frequency in an infected individual can be dominant (represented in the consensus sequence) in subsequent variants of interest or variants of concern. Here we briefly review the main conclusions of our work on mutant spectrum characterization of hepatitis C virus (HCV) and SARS-CoV-2 at the nucleotide and amino acid levels and address the following two new questions derived from previous results: (i) how is the SARS-CoV-2 mutant and deletion spectrum composition in diagnostic samples, when examined at progressively lower cut-off mutant frequency values in ultra-deep sequencing; (ii) how the frequency distribution of minority amino acid substitutions in SARS-CoV-2 compares with that of HCV sampled also from infected patients. The main conclusions are the following: (i) the number of different mutations found at low frequency in SARS-CoV-2 mutant spectra increases dramatically (50- to 100-fold) as the cut-off frequency for mutation detection is lowered from 0.5% to 0.1%, and (ii) that, contrary to HCV, SARS-CoV-2 mutant spectra exhibit a deficit of intermediate frequency amino acid substitutions. The possible origin and implications of mutant spectrum differences among RNA viruses are discussed.

SARS-CoV-2 Point Mutation and Deletion Spectra and Their Association with Different Disease Outcomes.

Mutant spectra of RNA viruses are important to understand viral pathogenesis and response to selective pressures. There is a need to characterize the complexity of mutant spectra in coronaviruses sampled from infected patients. In particular, the possible relationship between SARS-CoV-2 mutant spectrum complexity and disease associations has not been established. In the present study, we report an ultradeep sequencing (UDS) analysis of the mutant spectrum of amplicons from the nsp12 (polymerase)- and spike (S)-coding regions of 30 nasopharyngeal isolates (diagnostic samples) of SARS-CoV-2 of the first COVID-19 pandemic wave (Madrid, Spain, April 2020) classified according to the severity of ensuing COVID-19. Low-frequency mutations and deletions, counted relative to the consensus sequence of the corresponding isolate, were overwhelmingly abundant. We show that the average number of different point mutations, mutations per haplotype, and several diversity indices was significantly higher in SARS-CoV-2 isolated from patients who developed mild disease than in those associated with moderate or severe disease (exitus). No such bias was observed with RNA deletions. Location of amino acid substitutions in the three-dimensional structures of nsp12 (polymerase) and S suggest significant structural or functional effects. Thus, patients who develop mild symptoms may be a richer source of genetic variants of SARS-CoV-2 than patients with moderate or severe COVID-19. IMPORTANCE The study shows that mutant spectra of SARS-CoV-2 from diagnostic samples differ in point mutation abundance and complexity and that significantly larger values were observed in virus from patients who developed mild COVID-19 symptoms. Mutant spectrum complexity is not a uniform trait among isolates. The nature and location of low-frequency amino acid substitutions present in mutant spectra anticipate great potential for phenotypic diversification of SARS-CoV-2.

Importance of antibiotic penetration in the antimicrobial resistance of biofilm formed by non-pigmented rapidly growing mycobacteria against amikacin, ciprofloxacin and clarithromycin.

OBJECTIVES: To study the resistance of biofilms developed by non-pigmented rapidly growing mycobacteria (NPRGM) against amikacin, ciprofloxacin and clarithromycin in an in vitro model using clinical strains of different species. DESIGN: Antimicrobial susceptibilities of different clinical strains of Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium peregrinum, Mycobacterium mucogenicum and Mycobacterium mageritense using conventional techniques were measured. Biofilm resistance was measured by using the sandwich technique developed by Anderl et al. using a concentration of antibiotic of 50mg/L. Penetration of antibiotics through biofilm was measured using the same technique with minimal modifications. RESULTS: NPRGM biofilms showed drug resistance (percentages of viable bacteria >1% of those of controls) against antibiotics that are commonly used for the treatment of infections caused by these organisms, although there are intraspecies differences between strains. We have detected differences in antibiotic penetration through biofilms with an important permeability barrier for ciprofloxacin. However, other mechanisms must be probably more important to explain the antimicrobial resistance of NPRGM biofilm. CONCLUSIONS: Biofilms formed by NPRGM are resistant to amikacin, ciprofloxacin and clarithromycin. As no resistance differences between the tested antibiotics have been observed, it is likely that biofilm permeability of antibiotics is of low importance for antimicrobial resistance of biofilms.

High SARS-CoV-2 viral load is associated with a worse clinical outcome of COVID-19 disease.

COVID-19 severity and progression are determined by several host and virological factors that may influence the final outcome of SARS-CoV-2-infected patients. The objective of this work was to determine a possible association between viral load, obtained from nasopharyngeal swabs, and the severity of the infection in a cohort of 448 SARS-CoV-2-infected patients from a hospital in Madrid during the first outbreak of the pandemic in Spain. To perform this, we clinically classified patients as mild, moderate and severe COVID-19 according to a number of clinical parameters such as hospitalization requirement, need of oxygen therapy, admission to intensive care units and/or death. Also, Ct values were determined using SARS-CoV-2-specific oligonucleotides directed to ORF1ab. Here we report a statistically significant association between viral load and disease severity, a high viral load being associated with worse clinical prognosis, independently of several previously identified risk factors such as age, sex, hypertension, cardiovascular disease, diabetes, obesity and lung disease (asthma and chronic obstructive pulmonary disease). The data presented here reinforce viral load as a potential biomarker for predicting disease severity in SARS-CoV-2-infected patients. It is also an important parameter in viral evolution since it relates to the numbers and types of variant genomes present in a viral population, a potential determinant of disease progression.

COVID-19 in people living with HIV: A multicenter case-series study.

OBJECTIVES: Information on how COVID-19 affects people living with HIV (PLHIV) remains scarce. METHODS: An observational study was conducted in four public hospitals in Madrid. All HIV patients with confirmed or suspected COVID-19 were included and compared with COVID-19 patients without HIV infection. RESULTS: Sixty-three patients with HIV infection and confirmed or suspected COVID-19 were analyzed. The median age was 46 years (IQR: 37-56 years), and 88.9% were men. The median duration of HIV infection was 10.8 years (IQR: 6.5-16.8 years), and 96.8% were on antiretroviral therapy. 84.1% had previous comorbidities. The most common symptoms were fever (66.1%), cough (66.1%) and dyspnea (46.8%). Pneumonia was found in 47.5%, 28.6% of patients had severe disease, and 32.3% were admitted to hospital. The ICU admission rate and the mortality rate were both 3.17%. A significant association was observed between age, arterial hypertension, overweight, and diabetes mellitus and the severity of COVID-19. No association was observed between HIV-related factors and the severity of COVID-19. The rate of COVID-19 in HIV-patients was 1.68%. Similar hospitalization (31.74% vs 32.57%) and ICU admission (3.17% vs 2%) rates were observed with non-HIV infected patients. A lower mortality rate during hospitalization (10% vs 21.37%) and a lower global mortality rate (3.17% vs 6.96%) were also observed. CONCLUSIONS: Established poor prognostic factors for COVID-19 patients, such as age and comorbidities, remain the main determinants for PLHIV. Neither the HIV severity nor the type of ARV treatment seem to influence the outcome of COVID-19. Large prospective cohorts are needed in order to establish the differences between HIV-positive and HIV-negative patients.

Statin use is associated with reduced mortality after respiratory viral infection.

BACKGROUND: Several studies suggest that statins, besides reducing cardiovascular disease, have anti-inflammatory properties which might provide a benefit in downregulating the immune response after a respiratory viral infection (RVI) and, hence, decreasing subsequent complications. We aim to analyse the effect of statins on mortality after RVI. METHODS: A single-centre, observational and retrospective study was carried out including all adult patients with a RVI confirmed by PCR tests from October 2, 2017 to May 20, 2018. Patients were divided between statin users and non-statin users and followed-up for 1 year, and all causes of death were recorded. In order to analyse the effect of statin treatment on mortality after RVI we planned two different approaches, a multivariate Cox regression model with the overall population and a univariate Cox model with a propensity-score matched population. RESULTS: We included 448 patients, 154 (34.4%) of whom were under statin treatment. Statin users had a worse clinical profile (older population with more comorbidities). During the 1-year follow-up, 67 patients died, 17 (11.0%) in the statin group and 50 (17.1%) in the non-statin group. Multivariate Cox analysis showed that statins were associated with mortality benefit (HR 0.47, 95% CI 0.26-0.83; p=0.01). In a matched population (101 statins users and 101 non-statins users) statins also remained associated with mortality benefit (HR 0.32, 95% CI 0.14-0.72; p=0.006). Differences were mainly driven by non-cardiovascular mortality (HR 0.31, 95% CI 0.13-0.73; p=0.004). CONCLUSIONS: Chronic statin treatment was associated with reduced 1-year mortality in patients with laboratory-confirmed RVI. Further studies are needed to determine the exact role of statin therapy after RVI.

Compassionate use of tocilizumab in severe SARS-CoV2 pneumonia.

INTRODUCTION: Tocilizumab (TCZ) is an interleukin-6 receptor antagonist, which has been used for the treatment of severe SARS-CoV-2 pneumonia (SSP), which aims to ameliorate the cytokine release syndrome (CRS) induced acute respiratory distress syndrome (ARDS). However, there are no consistent data about who might benefit most from it. METHODS: We administered TCZ on a compassionate-use basis to patients with SSP who were hospitalized (excluding intensive care and intubated cases) and who required oxygen support to have a saturation >93%. The primary endpoint was intubation or death after 24 h of its administration. Patients received at least one dose of 400 mg intravenous TCZ from March 8, 2020 to April 20, 2020. RESULTS: A total of 207 patients were studied and 186 analyzed. The mean age was 65 years and 68% were male patients. A coexisting condition was present in 68% of cases. Prognostic factors of death were older age, higher IL-6, d-dimer and high-sensitivity C-reactive protein (HSCRP), lower total lymphocytes, and severe disease that requires additional oxygen support. The primary endpoint (intubation or death) was significantly worst (37% vs 13%, p < 0·001) in those receiving the drug when the oxygen support was high (FiO2 >0.5%). CONCLUSIONS: TCZ is well tolerated in patients with SSP, but it has a limited effect on the evolution of cases with high oxygen support needs.

Biofilm development by clinical isolates of Staphylococcus spp. from retrieved orthopedic prostheses.

BACKGROUND: Biofilms are considered the key factor in the development of implant-related infections. However, only a few reports have dealt with the ability of organisms isolated from such infections to develop biofilms in vitro. METHODS: We evaluated different phenotypic techniques (2 microtiter plate assays and confocal laser scanning microscopy (CLSM) and genotypic techniques (detection of the ica operon) related to biofilm development by clinical isolates of Staphylococcus spp. RESULTS: All 26 strains tested (from 23 specimens) were biofilm producers. Stepanovic test detected biofilm formation in 85% of the strains, microtiter plate assay in 65%, and CLSM in 39%. The ica operon was detected in 73% of all strains (all 13 S. aureus strains and 6 of the 13 coagulase-negative Staphylococcus strains). 7 ica-negative strains were biofilm-positive by phenotypic methods. INTERPRETATION: The detection of ica genes could not be related to the phenotypic ability of the strains to develop a biofilm in vitro, so both studies (genetic and phenotypic) are required for a better evaluation of the biofilm-producing ability of clinical strains of Staphylococcus isolated from orthopedic infections.