Performing Gram stain directly on catheter tips: assessment of the quality of the observation process.

A previous study performed in our institution showed that catheter tip (CT) staining by combining acridine orange and Gram stain (GS) before culture anticipated catheter colonization with exhaustive and careful observation by a highly trained technician. Our objective was to assess the validity values of GS without acridine orange on an external smear of CT for predicting catheter colonization and catheter-related bloodstream infection (C-RBSI). We compared different periods of observation and the results of two technicians with different levels of professional experience. Over a 5-month period, the roll-plate technique was preceded by direct GS of all CTs sent to the microbiology laboratory. The reading was taken at ×100 by two observers with different skill levels. Each observer performed a routine examination (3 min along three longitudinal lines) and an exhaustive examination (5 min along five longitudinal lines). The presence of at least one cell was considered positive. All slides were read before culture results were known. We included a total of 271 CTs from 209 patients. The prevalence of catheter colonization and C-RBSI was 16.2 % and 5.1 %, respectively. Routine and exhaustive examinations revealed only 29.5 % and 40.9 % of colonized catheters, respectively (p < 0.001). In contrast, they revealed high negative predictive values for C-RBSI (96.5 % and 96.3 %, respectively). Our study shows that the yield of GS performed directly on CTs is greater when staining is performed exhaustively. However, the decision to implement this approach in daily routine will depend on the prevalence rate of catheter colonization at each institution.

Uncommon aetiological agents of catheter-related bloodstream infections.

The clinical and microbiological characteristics of catheter-related bloodstream infection (CR-BSI) due to uncommon microorganisms was assessed in a retrospective case-control study over a 9-year period in a tertiary teaching hospital. Uncommon microorganisms were defined as those representing <0·5% of all CR-BSI. Diagnosis of CR-BSI required that the same microorganism was grown from at least one peripheral venous blood culture and a catheter tip culture. Thirty-one episodes of CR-BSI were identified due to 13 different genera and these accounted for 2·3% of all CR-BSI in the hospital. Although these infections were not associated with increased mortality, they occurred in patients with more severe underlying conditions who were receiving prolonged antibiotic therapy.

The risk of catheter-related bloodstream infection after withdrawal of colonized catheters is low.

Most episodes of catheter-related bloodstream infection (C-RBSI) are documented before or at the time of catheter withdrawal. The risk of C-RBSI in the period after removing a colonized catheter in patients without bacteremia (late C-RBSI) is unknown. We assessed the risk of developing a late C-RBSI episode in an unselected population with positive catheter tip cultures and analyzed associated risk factors. We analyzed retrospectively all colonized catheter tips between 2003 and 2010 and matched them with blood cultures. C-RBSI episodes were classified as early C-RBSI (positive blood cultures were obtained ≤24 h after catheter withdrawal) or late C-RBSI (positive blood cultures were obtained ≥24 h after catheter withdrawal). We analyzed the risk factors associated with late C-RBSI episodes by comparison with a selected group of early C-RBSI episodes. We collected a total of 17,981 catheter tips: 4,533 (25.2 %) were colonized. Of them, 1,063 (23.5 %) were associated to early C-RBSI episodes and from the remaining 3,470, only 143 (4.1 %) were associated to late C-RBSI episodes. Then, they corresponded to 11.9 % of the total 1,206 C-RBSI episodes. After comparing early and late C-RBSI episodes, we found that late C-RBSI was significantly associated with the presence of methicillin-resistant Staphylococcus aureus (MRSA, p = 0.028) and with higher mortality (p = 0.030). According to our data, patients with colonized catheter tips had a 4.1 % risk of developing late C-RBSI, which was associated with higher crude mortality.

Is catheter-related candidemia a polyclonal infection?

Diagnosis of catheter-related candidemia (CRC) requires the simultaneous isolation of Candida spp. from both blood and catheter samples. We previously observed that in most CRC cases, the genotype of the yeast found in catheter samples is also recovered from blood. However, it is not clear whether CRC is a polyclonal infection. We prospectively studied 20 patients with CRC caused by Candida albicans, C. parapsilosis, or C. glabrata to analyze whether their infections were polyclonal. As many as 10 colonies per sample (n = 475) isolated from blood (n = 220) and catheter (n = 255) specimens were studied using species-specific microsatellite markers. Genotyping always revealed matches between the Candida spp. from blood and catheter samples. However, 15% of patients had a polyclonal pattern of infection or catheter colonization that was species specific. An additional genotype was found exclusively in the catheters of two patients infected with C. albicans, whereas an additional genotype was noted in the blood culture of a patient infected with C. parapsilosis. Considering only the presence of different genotypes in blood samples, 5% of patients had polyclonal infections. We conclude that most cases of CRC are caused by a single genotype.

Value of superficial cultures for prediction of catheter-related bloodstream infection in long-term catheters: a prospective study.

Cultures taken from the skin and from the hubs of short-term central venous catheters can help us to predict catheter-related bloodstream infections (C-RBSIs). The value of these cultures for such predictions has not been assessed in long-term catheters. Our objective was to assess the value of superficial cultures for the prediction of C-RBSI among patients with long-term catheters. Over a 2-year period, we prospectively obtained cultures from the skin overlying reservoir ports (group A) and from the skin insertion site and hubs of all tunneled catheters (group B). This routine was performed by vascular and interventional radiologists immediately before catheter removal (irrespective of the reason for withdrawal). Swabs were processed semiquantitatively. Catheter tips from both groups were cultured using Maki's semiquantitative technique and sonication. We also performed cultures of the reservoir ports at different sites. C-RBSI was defined as the isolation of the same species of microorganism(s) both in the colonized catheter and in at least 1 peripheral blood culture. We included 372 catheters (group A, 223; group B, 149) during the study period. The catheter colonization rate was 23.4% (87/372), and 28 patients had C-RBSI. Validity index values for the capacity of surface cultures to predict C-RBSI in groups A and B were, respectively, as follows: sensitivity, 23.5% and 45.5%; specificity, 59.7% and 63.0%; positive predictive value, 4.6% and 8.9%; and negative predictive value, 90.4% and 93.5%. Superficial cultures of patients with long-term catheters could help us to rule out the catheter as the portal of entry of bloodstream infections. Superficial cultures (from skin and hubs) proved to be a useful conservative diagnostic tool for ruling out C-RBSI among patients with long-term tunneled catheters and totally implantable venous access ports.

Use of universal 16S rRNA gene PCR as a diagnostic tool for venous access port-related bloodstream infections.

Amplification of the universal 16S rRNA gene using PCR has improved the diagnostic yield of microbiological samples. However, no data have been reported on the reliability of this technique with venous access ports (VAPs). We assessed the utility of 16S rRNA PCR for the prediction of VAP-related bloodstream infection (VAP-RBSI). During a 2-year period, we prospectively received all VAPs removed by interventional radiologists. PCR and conventional cultures were performed using samples from the different VAP sites. We compared the results of PCR with those of conventional culture for patients with confirmed VAP-RBSI. We collected 219 VAPs from 219 patients. Conventional VAP culture revealed 15 episodes of VAP-RBSI. PCR revealed a further 4 episodes in patients undergoing antibiotic therapy which would have gone undetected using conventional culture. Moreover, it had a negative predictive value of 97.8% for the prediction of VAP-RBSI when it was performed using biofilm from the internal surface of the port. In conclusion, universal 16S rRNA PCR performed with samples from the inside of VAPs proved to be a useful tool for the diagnosis of VAP-RBSI. It increased detection of VAP-RBSI episodes by 21.1% in patients undergoing antibiotic therapy whose episodes would have gone undetected using conventional culture. Therefore, we propose a new application of 16S rRNA PCR as a useful tool for the diagnosis of VAP-RBSI in patients receiving antibiotic therapy.

How should long-term tunneled central venous catheters be managed in microbiology laboratories in order to provide an accurate diagnosis of colonization?

Guidelines recommend the roll-plate technique for short-term central venous catheter (CVC) tip cultures. However, the issue of whether the roll-plate technique is better than the sonication method for long-term CVCs remains unresolved. In addition, no data are available for predicting the value of direct Gram staining in anticipating catheter colonization or catheter-related bloodstream infection (CRBSI) in these long-term CVCs. Our objectives were to compare the roll-plate technique and the sonication method and to define the validity values of Gram staining for the prediction of colonization and CRBSI in patients with long-term tunneled CVCs. During the study period, all tunneled CVCs removed at our institution were prospectively and routinely sent to the microbiology laboratory for Gram staining (first) and tip culture (the Maki technique and sonication, in a random order). We received 149 tunneled CVCs, 39 (26.2%) of which were colonized and 11 (7.4%) of which were associated with CRBSI. Overall, the roll-plate method detected 94.9% of the colonized catheters, whereas sonication detected only 43.6% (P < 0.001). The validity values of Gram staining for the detection of colonization and CRBSI were as follows: a sensitivity of 35.9% to 60.0%, a specificity of 100% to 94.2%, a positive predictive value of 100% to 42.9%, and a negative predictive value of 81.5% to 97.0%. The roll-plate technique proved to be better than sonication for the detection of bacteria in long-term tunneled CVCs. Gram staining of the tips of tunneled CVCs can anticipate a positive culture and rule out CRBSI. In our opinion, direct Gram staining should be incorporated into routine microbiological assessments of long-term catheter tips.

Differential time to positivity (DTTP) for the diagnosis of catheter-related bloodstream infection: do we need to obtain one or more peripheral vein blood cultures?

The ideal number of blood samples to be obtained from peripheral veins (PVs) when differential time to positivity (DTTP) is being performed is an unresolved issue and most institutions obtain a single set. Our objective was to assess the number of proven central line-associated bloodstream infection (CLABSI) episodes that would have been recovered if blood had been cultured from one or two PVs. We performed a retrospective study in patients with proven CLABSI in which catheter lumens and two or more PV blood cultures were taken simultaneously. We calculated the number of episodes that would have been recovered if the culture of one or more PV blood cultures had been artificially eliminated. During a period of 4 years, we collected 60 episodes of proven CLABSI. Overall, if one PV culture had been eliminated in patients with two or three PV blood cultures, we would have documented 91.8% (p=0.362) and 96.9% (p>0.999) of episodes, respectively. If we had eliminated two PV blood cultures in patients with three PV blood cultures, 90.8% (p>0.999) of episodes would have been documented. When performing the DTTP technique to confirm CLABSI, a single paired PV blood culture was not associated with a significant number of missed CLABSI episodes.

Impact on knowledge and practice of an intervention to control catheter infection in the ICU.

Information on the impact of care bundles has been mainly acquired in adult intensive care units (ICUs). However, specific data for educational programs are scarce. Our objective was to analyze the impact of an educational program on the knowledge and prevention of catheter-related bloodstream infection (CRBSI) in two pediatric intensive care units (P-ICUs). A prospective study was carried out at a large teaching institution in Madrid, Spain. Healthcare workers' (HCWs) knowledge of guidelines for the prevention of CRBSI was assessed before and after the educational program using a questionnaire covering 12 issues. A 20-min program was offered to all HCWs on each ICU shift. The incidence density of CRBSI was assessed before, during, and after the educational program. A total of 174 questionnaires were completed by HCWs from both the neonatal ICU (N-ICU) and the P-ICU before the intervention and 54 were completed after the intervention (120 participants were not present during this period). The incidence density of CRBSI before, during, and after the intervention was 6.2, 5.2, and 9.3 in the N-ICU and 2.2, 3.1, and 2.9 in the P-ICU (p > 0.05). A single 20-min educational intervention on the prevention of CRBSI significantly improved HCWs' knowledge, but was not enough to reduce the incidence density of CRBSI.

Vaginal neutrophil infiltration is contingent on ovarian cycle phase and independent of pathogen infection.

The mucosa of the female reproductive tract must reconcile the presence of commensal microbiota and the transit of exogenous spermatozoa with the elimination of sexually transmitted pathogens. In the vagina, neutrophils are the principal cellular arm of innate immunity and constitute the first line of protection in response to infections or injury. Neutrophils are absent from the vaginal lumen during the ovulatory phase, probably to allow sperm to fertilize; however, the mechanisms that regulate neutrophil influx to the vagina in response to aggressions remain controversial. We have used mouse inseminations and infections of Neisseria gonorrhoeae, Candida albicans, Trichomonas vaginalis, and HSV-2 models. We demonstrate that neutrophil infiltration of the vaginal mucosa is distinctively contingent on the ovarian cycle phase and independent of the sperm and pathogen challenge, probably to prevent sperm from being attacked by neutrophils. Neutrophils extravasation is a multi-step cascade of events, which includes their adhesion through selectins (E, P and L) and integrins of the endothelial cells. We have discovered that cervical endothelial cells expressed selectin-E (SELE, CD62E) to favor neutrophils recruitment and estradiol down-regulated SELE expression during ovulation, which impaired neutrophil transendothelial migration and orchestrated sperm tolerance. Progesterone up-regulated SELE to restore surveillance after ovulation.

Surveillance of Chagas disease in pregnant women in Madrid, Spain, from 2008 to 2010.

One of the most important modes of transmission of Trypanosoma cruzi infection in areas where it is not endemic is vertical transmission: from mother to child. The objective of this report is to assess the efficacy of different programmes of serological screening to monitor infection with T. cruzi in pregnant Latin American women living in Madrid (Spain). To achieve this, a retrospective study was undertaken from January 2008 to December 2010 in seven hospitals in the Autonomous Community of Madrid. Serological screening programmes were classified in two main strategies: a selective one (pregnant women from Bolivia) and a universal one (pregnant women from Latin America). A total of 3,839 pregnant women were tested and the overall prevalence was 3.96%. The rate of congenital transmission was 2.6%. The current monitoring programmes have variable coverage ranging between 26% (selective screening) and 100% (universal screening). Monitoring of pregnant women from Latin America only reaches full coverage if universal screening of pregnant women is carried out at any moment of pregnancy, including at delivery. A common national regulation is necessary in order to ensure homogenous implementation of screening.

Application of real-time PCR for the differentiation of Entamoeba histolytica and E. dispar in cyst-positive faecal samples from 130 immigrants living in Spain.

In an effort to improve the diagnosis of intestinal amoebiasis, a real-time PCR has been used for the detection and differentiation of Entamoeba histolytica and E. dispar infections in African or South American immigrants who live in Spain. Faecal samples from all of the 130 subjects had apparently been found to contain E. histolytica/E. dispar cysts by microscopical examination. Using the real-time PCR, E. histolytica DNA was detected in faecal samples from only 10 (7.7%) of the immigrants, with E. dispar DNA detected in the samples from another 117 (90.0%) of the subjects. The use of such PCR in the routine investigation of patients found positive for E. histolytica/E. dispar cysts (by microscopy) is recommended, especially in non-endemic areas.

Tsukamurella: a cause of catheter-related bloodstream infections.

Tsukamurellae are strictly aerobic Gram-positive rods that can be easily misidentified as Corynebacterium species, Rhodococcus species, Nocardia species, Mycobacterium species, or other Gram-positive aerobic rods. They have been uncommonly reported as a cause of different human infections, including bloodstream infections. We describe 2 new cases of catheter-related bloodstream infections (CR-BSI) caused by Tsukamurella species and review 12 similar cases reported in the literature. Conventional procedures have often misidentified Tsukamurella species as other aerobic Gram-positive rods. This misidentification could be avoided using genotyping. All cases ultimately required the withdrawal of the infected line. The literature provides no firm conclusions regarding ideal choice or duration of antimicrobial therapy for this infection. Tsukamurella species should be added to the list of agents able to produce CR-BSI. Genotypic methods such as PCR 16S rRNA can allow a reliable identification at the genus level of Tsukamurella strains faster than a combination of conventional phenotypic methods.

Identification of Nocardia species from clinical isolates using MALDI-TOF mass spectrometry.

The identification of Nocardia isolates still represents a challenge for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) despite its acceptance for most bacterial and fungal isolates. In this study we evaluate the identification of Nocardia isolates using direct spotting and an updated database. Overall, 82 Nocardia isolates belonging to 13 species were identified by DNA sequence analysis of the 16S rRNA and secA1 genes. Nine of these well-characterized isolates from 6 Nocardia species were used to create an in-house library. The remaining 73 isolates were directly spotted on the target plate and on-plate protein extraction was performed. The protein spectra obtained were analyzed by MALDI-TOF MS using the BDAL database (Bruker Daltonics) updated with 6,903 MSPs or the combination of this commercial database and our in-house library. As a result, the use of the commercial database alone and in combination with the in-house library yielded 94.5% and 95.9% of correct species-level identifications, respectively, No isolate was misidentified at the genus level with either database. Besides, the use of the in-house library allowed the species-level identification of a N. otitidiscaviarum isolate that could only be identified at the genus-level with a score value <1.6 using the commercial database. In conclusion, the implementation of the direct spotting method and the in-house database provided a high rate of correct species assignment of Nocardia isolates despite the low number of isolates added. Further addition of well-characterized Nocardia isolates may ensure the rapid, accurate and inexpensive identification of most isolates encountered in the routine of the microbiology laboratory.

Nationwide study on peripheral-venous-catheter-associated-bloodstream infections in internal medicine departments.

BACKGROUND: The use of peripheral venous catheters (PVCs) has increased outside intensive care units, as has the rate of PVC-associated-bloodstream infection (PVC-BSI). PVCs are widely used in internal medicine departments (IMDs), but data on the incidence of PVC-BSI and its characteristics in IMDs are scarce. AIM: To assess the incidence of PVC-BSI episodes detected in IMDs in Spain. METHODS: A one-year multi-centre prospective observational cohort study in 14 Spanish IMDs was undertaken. Adult patients admitted with at least one PVC and bacteraemia were included in the study. Demographic and clinical data were provided by local coordinators. FINDINGS: Seventy episodes of PVC-BSI were recorded, representing an overall rate of 1.64 PVC-BSI episodes/1000 IMD admissions. The mean age of patients was 67.44 (standard deviation 16.72) years. It was estimated that 25.7% of PVCs were no longer necessary. Staphylococcus aureus was the most frequently isolated micro-organism (41.7%). Phlebitis was clinically evident in 44 (62.9%) episodes, and proved to be an independent predictor of catheter insertion in emergency departments (odds ratio 5.44). The crude and attributable mortality rates were 12.9% and 5.7%, respectively. CONCLUSIONS: PVCs carry a significant risk for bacteraemia in Spanish IMDs. Phlebitis is not always clinically evident in patients with bacteraemia in this population. The study findings support the need for educational and interventional preventive measures in both IMDs and emergency departments to reduce the rate of PVC-BSI and associated comorbidities, and costs.

Slicing silicone neonatal vascular catheter tips improves colonization detection by the roll-plate technique.

OBJECTIVE: Silicone neonatal peripherally inserted central catheters (SN-PICCs) are a common cause of catheter-related bloodstream infection (C-RBSI) in neonates. Our objective was to compare the yield of traditional roll-plate technique (TRP), roll-plate after slicing (RPS), and sonication after slicing (SS) for the detection of colonization and C-RBSI in SN-PICCs. METHODS: We prospectively cultured tips from SN-PICCs withdrawn from paediatric patients admitted to our institution with suspicion of infection. We first cultured the catheter tip using TRP and then divided the catheter into two segments. RPS was performed by longitudinally slicing one segment and the fragments were cultured. SS was performed by transversally slicing the other segment followed by culture after sonication. We calculated the validity values of each technique individually by comparing them with the diagnostic standard of colonization and C-RBSI. RESULTS: We included 162 SN-PICCs, 46 of which were colonized. Sensitivity rates for colonization and C-RBSI with TRP, RPS and SS were, respectively, 71.7%, 80.4% and 67.4%; and 74.2%, 90.3% and 77.4%. CONCLUSION: Catheter slicing should be performed before the roll-plate technique to ensure optimal diagnosis on SN-PICCs.

Improved method for the detection of catheter colonization and catheter-related bacteremia in newborns.

Accurate diagnosis of catheter-related bloodstream infection (CRBSI) is mandatory for hospital infection control. Peripherally inserted central venous catheters (PICCs) are widely used in intensive care units, but studies about procedures for detection of colonization are scarce in neonates. We sequentially processed 372 PICCs by 2 methods, first by the standard roll-plate (RP) technique and then by rubbing catheters on a blood agar plate after being longitudinally split (LS). With both techniques, we detected 133 colonized PICCs. Ninety-four events of CRBSI were diagnosed. The sensitivity, specificity, positive predictive value, and negative predictive value for detection of CRBSI were 58.5%, 92.8%, 73.3%, and 86.9%, respectively, for RP technique and 96.8%, 88.5%, 74.0%, and 98.8%, respectively, for LS technique. The LS technique increased the proportion of detected CRBSI by 38.3%. Neonatal PICC tips should be cultured after cutting them open. This technique is simple and sensitive to detect catheter colonization and also to diagnose CRBSI.

The misleading effect of serum galactomannan testing in high-risk haematology patients receiving prophylaxis with micafungin.

OBJECTIVES: To investigate the performance of the routine serum galactomannan (sGM) assay in the diagnosis of invasive aspergillosis (IA) in high-risk haematology patients receiving prophylaxis with micafungin. METHODS: Retrospective study including all haematological patients who received prophylaxis with micafungin during high-risk IA episodes (neutropenic patients after chemotherapy for acute myeloid leukaemia/myelodysplastic syndrome; allogeneic haematopoietic stem-cell transplantation during early neutropenic phase or graft-versus-host disease requiring high prednisone doses) and for whom at least one sGM result was available. Episodes were classified as follows: true-positive (positive GM in the context of IA), false-positive (positive GM result in patients who had no evidence of IA), true-negative (negative GM test results and no IA), or false-negative (negative GM test in the context of IA). Non-evaluable patients were excluded. RESULTS: Among 146 evaluable episodes, four were true-positive in the context of probable breakthrough IA (incidence of breakthrough IA, 2.7%); 111/146 high-risk episodes (76%) were considered true-negative and 31/146 (21.2%) were considered false-positive. No false-negative episodes were detected. All but one of the false-positive episodes were detected in surveillance GM tests, leading to high-resolution CT scans in eight cases (8/31; 25.8%), all of which were negative. The positive predictive and negative predictive values of sGM for surveillance and diagnostic approaches were 3.2% (1/31) and 100% (110/110) and 75% (3/4) and 100% (1/1), respectively. CONCLUSIONS: Surveillance of asymptomatic patients receiving prophylaxis with micafungin using sGM is unnecessary, because the results are either negative or false-positive. However, sGM remains useful in the diagnosis of breakthrough IA in symptomatic patients during prophylaxis.

Nationwide study on the use of intravascular catheters in internal medicine departments.

BACKGROUND: The use of intravascular catheters (IVCs) in intensive care units (ICUs) has been well assessed in recent years. However, a high proportion of these devices are placed in patients outside the ICU, particularly in internal medicine departments (IMDs), where data on the quality of care are scarce. AIM: To assess the use and management of IVCs in IMDs in Spain. METHODS: We performed a point prevalence study of all adult inpatients on 47 IMDs from hospitals of different sizes on one day in June 2013. A local co-ordinator was appointed to assess patients and collect data from each site. FINDINGS: Out of the 2080 adult patients hospitalized on the study day, 1703 (81.9%) had one or more IVCs (95.4% of which were peripheral devices). Infection was detected at the insertion site in 92 catheters (5.0%); 87 patients (5.2%) had signs of sepsis, but only one case was considered to be catheter-related. The local co-ordinators estimated that 19% of the catheters in place were no longer necessary. A daily record of the need for a catheter was available in only 40.6% of cases. CONCLUSION: Our study shows clear opportunities for improvement regarding catheter use and care in Spanish IMDs. Strategies similar to those applied in ICUs should be implemented in IMDs.