Blood Culture Procedures Research Articles

Blood culture-free ultra-rapid antimicrobial susceptibility testing.

Treatment assessment and patient outcome for sepsis depend predominantly on the timely administration of appropriate antibiotics1-3. However, the clinical protocols used to stratify and select patient-specific optimal therapy are extremely slow4. In particular, the major hurdle in performing rapid antimicrobial susceptibility testing (AST) remains in the lengthy blood culture procedure, which has long been considered unavoidable due to the limited number of pathogens present in the patient's blood. Here we describe an ultra-rapid AST method that bypasses the need for traditional blood culture, thereby demonstrating potential to reduce the turnaround time of reporting drug susceptibility profiles by more than 40-60 h compared with hospital AST workflows. Introducing a synthetic beta-2-glycoproteinI peptide, a broad range of microbial pathogens are selectively recovered from whole blood, subjected to species identification or instantly proliferated and phenotypically evaluated for various drug conditions using a low-inoculum AST chip. The platform was clinically evaluated by the enrolment of 190 hospitalized patients suspected of having infection, achieving 100% match in species identification. Among the eight positive cases, six clinical isolates were retrospectively tested for AST showing an overall categorical agreement of 94.90% with an average theoretical turnaround time of 13 ± 2.53 h starting from initial blood processing.

Time to Positivity of Blood Culture as a Predictor of Causative Pathogens and Survival in Neonatal Sepsis: A Retrospective Cohort Study from Indonesia.

In the blood culture procedure for neonatal sepsis, time to positivity (TTP) reflects the pathogenic bacterial load and the time required for empirical antibiotic regimen administration prior to definitive treatment. This study aims to identify the differences in TTP among causative pathogens and its predictive value for the overall survival of neonates with sepsis at a tertiary healthcare center in Indonesia. A retrospective cohort study was conducted from January 2020 to August 2022 at Dr. Soetomo General Hospital, Surabaya, Indonesia. Neonates with blood culture-proven neonatal sepsis were included in the analysis. TTP was defined as the time between the acceptance of a blood culture specimen from the neonatal intensive care unit and reports of positive culture growth by the laboratory. Across 125 cases, the median TTP was 58.1 hours (IQR = 24.48). Blood cultures were positive within 48 hours for 41.6% of cases, 72 hours for 86.4%, and 96 hours for 98.4%. A significantly shorter TTP was exhibited by the three major gram-negative organisms (Klebsiella pneumoniae,Acinetobacter baumannii,Enterobacter cloacae) compared to coagulase-negative Staphylococci. The neonatal sepsis mortality rate was 49.6% during the study period. In the Cox multivariate regression model, a shorter TTP was an independently predicted mortality in the entire cohort (hazard ratio (HR) = 0.985, 95% CI: 0.973-0.998) and the gram-negative sepsis cohort group (HR = 0.983, 95% CI: 0.968-0.999). TTP predicts different causative pathogens and the overall survival of neonatal sepsis cases at a tertiary healthcare facility in Indonesia.

Blood culture procedures and practices in the neonatal intensive care unit: A survey of a large multicenter collaborative in California.

To describe variation in blood culture practices in the neonatal intensive care unit (NICU). Survey of neonatal practitioners involved with blood culturing and NICU-level policy development. We included 28 NICUs in a large antimicrobial stewardship quality improvement program through the California Perinatal Quality Care Collaborative. Web-based survey of bedside blood culture practices and NICU- and laboratory-level practices. We evaluated adherence to recommended practices. Most NICUs did not have a procedural competency (54%), did not document the sample volume (75%), did not receive a culture contamination report (57%), and/or did not require reporting to the provider if <1 mL blood was obtained (64%). The skin asepsis procedure varied across NICUs. Only 71% had a written procedure, but ≥86% changed the needle and disinfected the bottle top prior to inoculation. More than one-fifth of NICUs draw a culture from an intravascular device only (if present). Of 13 modifiable practices related to culture and contamination, NICUs with nurse practitioners more frequently adopted >50% of practices, compared to units without (92% vs 50% of units; P < .02). In the NICU setting, recommended practices for blood culturing were not routinely performed.

Improving awareness of blood culture indication, procedure and resource Utilization at Armed Forces Hospital Jizan, Saudi Arabia

Blood culture contamination (BCC) is a significant quality and safety issue in hospitals, as it leads to increase in unnecessary testing, admissions, antibiotic exposure and cost. This study is the...

Identification of Peptoniphilus vaginalis-Like Bacteria, Peptoniphilus septimus sp. nov., From Blood Cultures in a Cervical Cancer Patient Receiving Chemotherapy: Case and Implications

A 39-year-old woman with a 3-year human papillomavirus (HPV) 18 infection history was admitted to the hospital for a 16-day history of vaginal bleeding after sex. She was diagnosed with cervical cancer based on the results of the electronic colposcopy, cervical cytology, microscopy, and magnetic resonance imaging (MRI). Then, she received chemotherapy, with paclitaxel 200 mg (day 1), cisplatin 75 mg (day 2), and bevacizumab 700 mg (day 3) twice with an interval of 27 days. During the examination for the diagnosis and treatment, many invasive operations, including removal of intrauterine device, colposcopy, and ureteral dilatation, were done. After that, the patient was discharged and entered the emergency department about 2.5 months later with a loss of consciousness probably caused by septic shock. The patient finally died of multiple organ failure and bacterial infection, although she has received antimicrobial therapy. The blood cultures showed a monobacterial infection with an anaerobic Gram-positive bacterial strain, designated as SAHP1. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI–TOF MS) indicated that the patient was infected with Peptoniphilus asaccharolyticus, while molecular analysis and genome-based taxonomy confirmed the infection with a novel Peptoniphilus species that has a close genetic relationship with Peptoniphilus vaginalis and proposed provisionally as Peptoniphilus septimus sp. nov., which may also act as a commensal of the human vagina. Genomic features of SAHP1 have been fully described, and comparative genomic analysis reveals the known prokaryote relative of Peptoniphilus septimus sp. nov. in the genus Peptoniphilus. The invasive operations on the genital tract during the diagnosis and treatment of the patient and the tumor tissue damage and bleeding may have a certain role in the bloodstream infection. This study casts a new light on the Peptoniphilus bacteria and prompts clinicians to include anaerobic blood cultures as part of their blood culture procedures, especially on patients with genital tract tumors. Furthermore, due to the incomplete database and unsatisfying resolution of the MALDI–TOF MS for Peptoniphilus species identification, molecular identification, especially whole-genome sequencing, is required for those initially identified as bacteria belonging to Peptoniphilus in the clinical laboratory.

Development and validation of case-finding algorithms to identify prosthetic joint infections after total knee arthroplasty in Veterans Health Administration data.

To determine the positive predictive values (PPVs) of ICD-9, ICD-10, and current procedural terminology (CPT)-based diagnostic coding algorithms to identify prosthetic joint infection (PJI) following knee arthroplasty (TKA) within the United States Veterans Health Administration. We identified patients with: (1) hospital discharge ICD-9 or ICD-10 diagnosis of PJI, (2) ICD-9, ICD-10, or CPT procedure code for TKA prior to PJI diagnosis, (3) CPT code for knee X-ray within ±90 days of the PJI diagnosis, and (4) at least 1 CPT code for arthrocentesis, arthrotomy, blood culture, or microbiologic procedure within ±90 days of the PJI diagnosis date. Separate samples of patients identified with the ICD-9 and ICD-10-based PJI diagnoses were obtained, stratified by TKA procedure volume at each medical center. Medical records of sampled patients were reviewed by infectious disease clinicians to adjudicate PJI events. The PPV (95% confidence interval [CI]) for the ICD-9 and ICD-10 PJI algorithms were calculated. Among a sample of 80 patients meeting the ICD-9 PJI algorithm, 60 (PPV 75.0%, [CI 64.1%-84.0%]) had confirmed PJI. Among 80 patients who met the ICD-10 PJI algorithm, 68 (PPV 85.0%, [CI 75.3%-92.0%]) had a confirmed diagnosis. An algorithm consisting of an ICD-9 or ICD-10 PJI diagnosis following a TKA code combined with CPT codes for a knee X-ray and either a relevant surgical procedure or microbiologic culture yielded a PPV of 75.0% (ICD-9) and 85.0% (ICD-10), for confirmed PJI events and could be considered for use in future pharmacoepidemiologic studies.

Improved blood culture workflow in the time to detection of microorganisms placing incubators systems outside of microbiology laboratory.

We analyzed the workflow of the blood culture procedure with one blood culture incubator in the microbiology laboratory, in comparison with the workflow with the incubators systems placing outside, and in a microbiology laboratory without 24-h staffing. We assessed the elapsed time (ET) and time-to-result (TTR) in the two laboratory workflows during 1month period in consecutive years. First period with one BACT/ALERT 3D module located in the microbiology laboratory (ML) (access 8 a.m. to 10 p.m.) and second period with three BACT/ALERT VIRTUO modules (one located in ML and two in the core sample laboratory, access 24h). The mean ET with BACT/ALERT 3D was 7.09 ± 6.15h and 1.32 ± 3.14h with BACT/ALERT VIRTUO. During the 8:00 a.m. to 10:00 p.m. shift, the average ETs were 3.54 ± 5.06 vs 1.59 ± 1.29h for the two time periods, respectively. Since the automated loading of bottles on the BACT/ALERT VIRTUO allows processing of blood cultures during the night shift, there was a significant reduction of time during the 10:00 p.m. to 8:00 a.m. shift, where the average ET was 10.52 ± 5.23 vs 1.00 ± 4.40h, respectively. The percentage of positivity in the first period was 9.03% and 11.18% in the second (p = 0.0003). The average TTR in the first period was 24.78 ± 15.9h and 16.85 ± 14.13h in the second (p < 0.0001). Easy 24-h access to blood culture incubators resulted in significant improvement in the workflow of blood culture, decreasing ET, and therefore decreasing the time to positivity and the efficiency of recovery.

Performance of Neonatal Intensive Care Unit Nurses in Blood Culture Procedure in Tabriz Hospitals in 2016

Background: Blood culture is a method in neonatal intensive care unit (NICU) and a gold standard for the diagnosis of infection that is essential for proper newborn management. The aim of this study was to compare the performance of nurses in the NICUs of Tabriz University of Medical Sciences using a standard sampling method. Methods: This descriptive cross-sectional study was performed on 90 nurses working in Al-Zahra and Taleghani ‎Educational and Medical Centers, and Tabriz Children Hospital using the convenience sampling method. The study tool included a two-part questionnaire in which the first part contained demographic characteristics of the infants and nurses and the service area. The second part included a checklist of the nurses' performance before and during ‎the blood culture procedure. Data analysis was performed using SPSS statistical software (version 21), as well as descriptive and analytical statistics. Results: Most nurses did not attend training courses for blood culture. The total performance scores before and during blood ‎culture sampling were ‎13.20‎‏±‏‎2.57 and ‎12.48‎‏±‏‎2‎.14‎, respectively.‎ Conclusion: Nurses are responsible for the correct sampling of blood culture and this procedure requires sufficient skill. Therefore, organizations must adapt their system to the standards in order to determine the benefits and disadvantages of the standards.

血液培養における汚染率をめぐる用語の混乱

The blood culture contamination rate is often used to validate specimen-collection procedures. CUMITECH has set its optimal target to be 2% to 3%. However, the term "contamination rate" has been defined in many ways, limiting its generalizability. The definitions used in earlier studies can be divided into two categories; definitions based on clinical judgements, and those based on preset rules. According to each principle, the equation must be composed of a defined numerator and denominator. The problem with clinical definitions is that the decision is inevitably subjective, and the process is too cumbersome. Also, if the number of positive cultures is used as the denominator, the value would be equivalent to the positive predictive value, given that contamination is regarded as a "positive case." Thus, the value would not be useful for validating a procedure. On the other hand, when the preset algorithm was adopted, true infection would, to some degree, inevitably be classified as contamination. Also, if the algorithm adopted the number of blood culture sets as the denominator and contamination was defined as the identification of 1 or more specified organisms in only 1 of multiple sets of blood cultures, its theoretical maximum value would not be 100%. This is a problem because the value is a mixture of several numbers with different scales. In other words, whether the blood cultures are collected once, twice, or thrice or more a day would affect the result. The study cited by CUMITECH aimed to evaluate the equivalence between the clinical definition and the laboratory definition with preset rules, rather than to establish a benchmark for the contamination rate. It is undesirable for the number to be perceived as a benchmark. "A Guide to Blood Culture" (2013) by the Japanese Society for Clinical Microbiology introduced a calculation for the contamination rate, but the definition of the term "number of specimens" in the formula is ambiguous. In addition, the references cited in the guide do not concern contamination and do not even mention the definition of contamination rate. Thus, it is impossible to confirm the definition. In view of the weaknesses of these previous works, we defined the contamination rate as a benchmark for the validation of blood culture procedures as follows. [number of series in which 1 or more specified organisms (*) were identified in only 1 of multiple sets of blood cultures]/[total number of multiple sets of blood cultures in the series] *coagulase-negative staphylococci, Propionibacterium acnes, Micrococcus spp., Viridans-group streptococci, Corynebacterium spp., and Bacillus spp., but not B. anthracis.

Using Lean Management to Reduce Blood Culture Contamination

Blood culture contamination (BCC) is a common and avoidable complication of patient care and incurs considerable cost. A quality improvement (QI) initiative was undertaken at a large Department of Veterans Affairs (VA) medical center to reduce the BCC rate. Lean management QI methods, including a rapid process improvement workshop (RPIW), were used to identify root causes of variation in blood culture procedures and countermeasures (potential improvement strategies) to address each problem were developed. BCC rates were collected for five and one quarter years, including the pre-RPIW (baseline) period, and changes in the contamination rates were calculated. The observed change in BCC rates was compared to a forecast of the pre-RPIW trend and estimated BCCs avoided. Results for the primary medical center were compared with those of a similarly complex VA medical center during the same time periods using difference-in-differences methodology. Qualitative assessment of the processes of care identified four root cause problems, each of which was addressed with countermeasures. The BCC rate at the primary medical center decreased significantly from the baseline period in each year of follow-up, improving from 4.2% in the 19-month baseline period to 2.8% in the last 12 months of follow-up (April 2013-March 2014), while changes from baseline in the BCC rate at the comparison site were significant in only one year of follow-up. An estimated 261 BCCs were avoided at the primary medical center in the follow-up period. The QI initiative was successful in reducing BCC rates and in producing continued improvement for nearly four years of follow-up. Further study will determine if these results are generalizable to other settings.

French national proficiency testing study for the standardisation of the CFU-GM progenitor assay

The Laboratory Controls Division of the French Health Product Safety Agency (ANSM) had organized annual external quality control (EQC) of Hematopoietic Stem Cells (HSC) preparations from 2000 to 2013. From this experience, it appeared that it was necessary to prepare new technical guidelines for the clonogenic assay. Indeed the present guidelines did not give sufficient tools of validation. The analysis of data collected from the EQC allowed to produce recommendations which have been placed in public inquiry for three months in 2013. In order to evaluate these ones, a Proficiency Testing Study was conducted with 29 control laboratories of HSC. To that end, samples of fresh cord blood, culture medium and cell culture procedure were sent to participants. Three conditions were proposed: seeding 200 cells CD34+ per dish (compulsory requirement) with initial cord blood then according to laboratories practices, seeding at another cell concentration and/or after erythrocyte removal. For the reference condition, coefficients of variation are inferior to 40%, while discrepancies vary much more for the two other conditions (with a maximum value of 116.6%). In order to carry on the assessment of erythrocyte removal procedures initiated during this study, we performed additional testing of sedimentation. They have shown that this simple and fast method can be used with reliability but only in intra-laboratory conditions. Alongside, comments received during the public inquiry were studied. The answers were documented by analyzing data from EQC that showed an improvement of reproducibility of results obtained in 2011-2012. Eventually, the recommendations were adjusted according to our studies and will be evaluated by the Committee of biological products of the French Pharmacopoeia. All our results showing that the clonogenic assay can be used reliably to evaluate the quality of hematopoietic grafts should allow to base the publication of these recommendations in early 2014.

Understanding Laboratory Methods and Their Impact on Antimicrobial Resistance Surveillance, at Muhimbili National Hospital, Dar es Salaam, Tanzania

The study sought to describe laboratory methods and blood culture procedures and their impact on antimicrobial resistance surveillance among nosocomial bacteria. We conducted a systematic audit of blood culture procedures and practices in the Department of Microbiology, Central Pathology Laboratory at Muhimbili National Hospital, between 19th and 23rd March 2012. A total of 25 -30 blood culture specimens were received each day as an indication of low volumes of blood culturing at this site. More blood culture requests came from the neonatal unit of the hospital, and were performed manually with high culture negative specimens. The laboratory performed antibiotic susceptibility testing as per the CLSI guidelines. No vancomycin resistance was ever reported at this site. All blood culture results were entered into the JEEVA laboratory information system, where results could be accessed by clinicians in the wards and data could be retrieved to assess patterns of antimicrobial resistance. Blood culture data entry system lacked quality control checks hence numerous errors and missing data were observed. Our results support the relevance of having improved laboratory procedures and good quality blood culture since surveillance of antimicrobial resistance primarily depends on good laboratory procedures, good quality and reliable blood culture data. This would essentially minimise imprecise estimates of rates of antimicrobial resistance at this hospital.

Compliance with Blood Volumes Collected for Blood Cultures between Physicians and Phlebotomists

Background: Blood culture is essential for the diagnosis and management of bloodstream infections. Blood volume is a key parameter determining the success of blood cultures. Studies comparing compliance between physicians and phlebotomists regarding optimal blood culture procedure are very rare in Korea. Methods: After educating physicians (interns) and phlebotomists about the correct procedure for blood culturing, the blood volumes of forty-three percent of randomly selected aerobic and anaerobic culture sets for adult patients (≥18 years old) were compared between these two groups over a period of three months. Physicians obtained blood from all admitted patients except those in the emergency department, where phlebotomists performed blood collection. Results: The numbers of blood culture sets requested during the study period were 3,238 and 2,136 for the physician and phlebotomist groups, respectively. The blood volumes of blood culture sets were significantly higher for the phlebotomists (16.7 mL) than for the physicians (9.2 mL). The positive rate of blood culture was also higher for the phlebotomist group (10.3% vs. 7.9%). The contamination rates (0.8%) were the same for both groups. Conclusion: Although the patients' medical conditions, antibiotics prescriptions, or duration of hospitalization may have affected the positive rate of blood cultures, this rate might also have been influenced by the blood volume. The compliance of phlebotomists was greater than that of physicians regarding the blood volume collected for blood cultures. (Ann Clin Microbiol 2013;16:81-86)

Blood Culture Procedures and Results in a Pediatric Hospital in La Paz, Bolivia: Opportunities for Improving Efficiency and Decreasing Cost

Blood Culture Procedures and Results in a Pediatric Hospital in La Paz, Bolivia: Opportunities for Improving Efficiency and Decreasing Cost

Approaches to Minimize Infection Risk in Blood Banking and Transfusion Practice

The use of blood donor history and state-of-the-art FDA-licensed serological and nucleic acid testing (NAT) assays have greatly reduced the "infectious window" for several transfusion-transmitted pathogens. Currently transmission of human immunodeficiency virus (HIV), Human T-cell Lymphotropic Virus (HTLV), hepatitis viruses and West Nile Virus are rare events. The seroprevalence of cytomegalovirus in the donor population is high and cytomegalovirus infection can cause significant complications for immunocompromised recipients of blood transfusion. Careful use of CMV seronegative blood resources and leukoreduction of blood products are able to prevent most CMV infections in these patients. Currently, bacterial contamination of platelet concentrates is the greatest remaining infectious disease risk in blood transfusion. Specialized donor collection procedures reduce the risk of bacterial contamination of blood products; blood culture and surrogate testing procedures are used to detect potential bacterially contaminated platelet products prior to transfusion. A rapid quantitative immunoassay is now available to test for the presence of lipotechoic acid and lipopolysaccharide bacterial products prior to platelet transfusion. Attention has now turned to emerging infectious diseases including variant Creutzfeldt-Jakob disease, dengue, babesiosis, Chagas' disease and malaria. Challenges are presented to identify and prevent transmission of these agents. Several methods are being used or in development to reduce infectivity of blood products, including solvent-detergent processing of plasma and nucleic acid cross-linking via photochemical reactions with methylene blue, riboflavin, psoralen and alkylating agents. Several opportunities exist to further improve blood safety through advances in infectious disease screening and pathogen inactivation methods.

A comparison of two blood culture procedures for the isolation of staphylococci in a paediatric intensive care unit

Blood culture results obtained between January 2000 and July 2003 were reviewed for 1360 patients in a paediatric intensive care unit (PICU). The BacT/Alert FA aerobic medium was used with a blood volume of 1.5 mL for the first 23 months, and the BacT/Alert PF paediatric medium was used with a 0.5-mL volume for the remaining 18 months. The isolation rates were similar during both periods (13.4% vs. 13.1%), and staphylococci were the most common isolates (72.8%). There was a shorter time to detection of staphylococci with the smaller-volume (PF) procedure, which thus seems suitable for use in the diagnosis of staphylococcal bacteraemia in the PICU.

NOR expression increases on metaphase chromosomes of down syndrome lymphocytes in concordance with mitogen concentration in culture medium

Regulation of nucleolus organizer region (NOR) expression in trisomy 21 (Down syndrome [DS]) cells is not fully explained. This work compared NOR expression on metaphase chromosomes in gradiently stimulated lymphocytes from DS patients with those from healthy controls. Conventional peripheral blood culture (72 h) and chromosomal preparation procedures were used except that blood samples from each individual were cultivated in the same but gradiently increasing concentrations (0.37, 0.75, 1.48, and 2.21 ml) of phytohemagglutinin (PHA) per 100 ml of medium. One hundred consecutive metaphases per concentration were analyzed for scoring the means of the active NORs bearing chromosomes (AgNOR+ chromosome) per individual and per concentration. In contrast to healthy controls (n=24), AgNOR+ chromosomal number in lymphocytes from 30 DS patients increased in concordance to the gradient of PHA concentration in the culture medium. DS lymphocytes do not downregulate their NOR expression in the limit of control cells. This in vitro result may serve as a clue for the explanation of the DS phenotype due to the wasted energy in producing unnecessary rRNA transcripts and AgNOR proteins in utero during organogenesis. These results also indicate that precautions must be used in routine work of NOR evaluation/interpretation in DS lymphocytes.

Detection of methicillin resistance in coagulase-negative staphylococci and in staphylococci directly from simulated blood cultures using the EVIGENE MRSA Detection Kit.

The EVIGENE MRSA Detection Kit was evaluated on coagulase-negative staphylococci (CoNS) from agar plates and on staphylococci directly from positive spiked blood cultures. For the CoNS study, a total of 242 isolates were tested, and of these 237 gave valid test results. For the 237 valid tests, all gave correct mecA classification. For the blood culture procedure, a collection of 51 mecA-positive Staphylococcus aureus, 21 mecA-negative S. aureus, 31 mecA-positive CoNS and 28 mecA-negative CoNS were used for the simulated blood cultures. For the S. aureus strains, all gave valid test results and correct mecA classification. One of the MRSA isolates gave a very faint nuc signal, and another four isolates gave results close to the cut-off of the kit; however, these were still clearly positive when read by the naked eye. For the CoNS isolates, 51 of the 59 strains gave valid results. All of these 51 strains gave correct mecA status. Thus the EVIGENE MRSA Detection Kit can provide fast and accurate determination of methicillin resistance in CoNS. This preliminary study of the blood culture procedure indicates that it is possible to achieve determination of methicillin resistance in staphylococci 8 h after positivity of the blood culture, making same-day detection of methicillin resistance possible.

Etiology of Community-acquired Pneumonia in Hospitalized Patients: A 3-Year Prospective Study in Japan

To compare the etiology of community-acquired pneumonia in Japan and Western countries, the causative pathogens were prospectively investigated in patients requiring hospitalization.Prospective study over a 3-year period.A community general hospital in Japan.Three hundred twenty-six episodes of community-acquired pneumonia in 318 patients admitted to the hospital between July 1994 and June 1997.The microbiological diagnosis was based on the results of quantitative sputum culture, blood culture, and other invasive procedures, including transthoracic needle aspiration or bronchoscopic examination. Serologic tests for Mycoplasma pneumoniae, Chlamydia spp, Legionella spp, and viruses were also routinely performed.Causative pathogens were identified in 199 episodes (61%). Streptococcus pneumoniae was the most common pathogen (23%), followed by Haemophilus influenzae (7.4%), M pneumoniae (4.9%), and Klebsiella pneumoniae (4.3%). The Streptococcus milleri group and Chlamydia pneumoniae were detected in 3.7 and 3.4% of the episodes, respectively. Pneumonia due to Legionella spp was recognized in only two patients.The etiology of community-acquired pneumonia in Japan did not differ markedly when compared with that of Western countries except for the low incidence of Legionella pneumonia. C pneumoniae and the S milleri group, which are emerging or newly recognized pathogens, were also significant causative microorganisms.

A rapid method for the diagnosis of Charcot Marie Tooth disease Type 1A by FISH

▼Charcot Marie Tooth disease Type 1A (CMT1A) is associated with a submicroscopic tandem duplication on the short arm of chromosome 17. The 1.5 Mb duplication contains the peripheral myelin protein 22 (PMP22) gene, the dosage of which plays a crucial role in the aetiology of the disorder. Diagnosis of CMT1A has until recently been limited to studies on Southern blots. Methods derived for fluorescence in situ hybridization (FISH) analysis have relied on traditional 72-h cytogenetic cultures of patient blood samples (Ref. 1) or on the development of more complex blood treatment protocols (Ref. 2). Metaphase chromosomes cannot be used because the duplication is tandem and unresolvable by FISH at metaphase level. We describe a rapid, reliable method producing large numbers of well-conserved nuclei, suitable for FISH, that fits easily into the routine procedures of a busy diagnostic cytogenetic laboratory. The main modification of standard blood culture procedures is the reduction of culture time to overnight, with no colchicine treatment. Stock culture medium can be used, containing phytohemagglutinin (PHA) if this is the way stock is usually prepared, since overnight culture is insufficient to stimulate nuclei past the G0/G1 stage of the cell cycle. This is important, for the replicated chromatids of late S or G2 frequently give double signals, potentially mimicking the CMT1A duplication. This method therefore obviates the need to co-hybridize an alternate-coloured control probe to check the replication status of the 17p segment. Blood cultures for diagnosis are set up the day before any normal harvest day. Any standard chromosome harvesting procedure that includes hypotonic treatment and acetic