Clinical and bacteriological substantiation of the use of cefoperazone/sulbactam in complex therapy of patients with pyo-destructive forms of lower respiratory tract infection (LRTI)]

Antibacterial therapy is the most important component of the complex management of pyo-destructive forms of LRTI. Since the microbial flora is rather variable and polymorphous, antibiotics used in the treatment of LRTI should be active against both aerobic (especially gramnegative) and anaerobic pathogens. The aim of the study was to prove, on the basis of the bacteriological and clinical findings, the validity of the use of cefoperazone/sulbactam (CS), the only inhibitor-protected cephalosporin, for the monotherapy of patients with LRTI. The trial enrolled 32 patients (29 males and 3 females) with various forms of LRTI, including 22 patients with destructive pneumonia, 8 patients with acute and chronic lung abscesses and 2 patients with lung gangrene. Complications of the main disease such as empyema, bronchopleural fistula, pyopneumothorax and hemoptysis in 63.5% of the patients were recorded. To verify the microbiological diagnoses, bacteriological assay of the sputum, endobronchial secretion or the contents of the abscess and pleural cavities was performed. The main component of the complex conservative treatment was the monotherapy with CS administered intravenously in an average daily dose of 5.9+/-1.59 g divided into 2 portions. The maximum daily dose for the patients with lung gangrene was 12 g. The bacteriological efficacy was evaluated by the ESCMID (1993) criteria. The clinicoroentgenologic efficacy was estimated by regression of the main signs of LRTI. The pathogens of LRTI were isolated and identified in 87.5% of the patients. Nonsporulating anaerobic bacteria such as Prevotella spp., Bacteroidesfragilis, Fusobacterium spp., Peptococcus spp. and Peptostreptococcus spp. were isolated from 24 (75%) of them. AD the anaerobic organisms proved to be susceptible to CS (100%). As for the aerobic organisms, 85.5% of them was susceptible to CS. The clinical effect of the antibacterial therapy in 29 (90.6%) patients was registered. In 20 patients (64.5%) both clinical and roentgenologic cure was shown. The lethal outcome in 1 patient (3.1%) was stated.     

Airway Interleukin-8 in Elderly Patients with Bacterial Lower Respiratory Tract Infections

We investigated the interleukin-8 (IL-8) levels and neutrophil numbers in the sputum of 9 elderly patients with lower respiratory tract infections, including Pseudomonas aeruginosa infection, before and after treatment with various antimicrobial agents. The IL-8 levels in sputum supernatants and the neutrophil numbers in sputum smears from 9 patients decreased significantly after the elimination of the causative respiratory pathogens. We also demonstrated that human recombinant IL-8 at a range of 6.25-25 ng/ml significantly enhanced opsonophagocytic killing of P. aeruginosa immunotype-1 strain by human neutrophils in the presence of a serotype-specific anti-lipopolysaccharide monoclonal antibody and fresh normal human serum. These data suggest that the level of IL-8 production in the airways of patients with lower respiratory tract infections is dependent on bacterial densities, and indicate the important role of IL-8 not only in neutrophil migration but also in opsonophagocytic killing of bacteria in the lower respiratory tract.     

Bacteriological Assessment of Healthcare-Associated Pneumonia Using a Clone Library Analysis

Background

The causative pathogens of healthcare-associated pneumonia (HCAP) remain controversial, and the use of conventional cultivation of sputum samples is occasionally inappropriate due to the potential for oral bacterial contamination. It is also sometimes difficult to determine whether methicillin-resistant Staphylococcus aureus (MRSA) is a true causative pathogen of HCAP.

Methods

We evaluated the bacterial diversity in bronchoalveolar lavage fluid (BALF) using molecular and cultivation methods in 82 HCAP patients. BALF specimens were obtained from the lesions of pneumonia using bronchoscopy. The bacterial flora was analyzed according to the clone library method using amplified fragments of the 16S ribosomal RNA gene with universal primers. In addition, sputum cultures and the above specimens were assessed.

Results

Eighty (97.6%) of the 82 BALF samples obtained from the patients with HCAP showed positive polymerase chain reaction results. The predominant phylotypes detected in the BALF in this study included bacteria common in cases of community- and hospital-acquired pneumonia. In addition, the phylotypes of streptococci and anaerobes were detected in 19 (23.2%) and 8 (9.8%) cases, respectively. In particular, phylotypes of streptococci were highly detected among the patients 75 of age or older. Staphylococcus aureus was cultured in 23 (28.0%) cases using conventional cultivation methods and detected in only 6 (7.3%) cases as predominant phylotypes according to the clone library method.

Conclusions

The clone library analysis of BALF in the HCAP patients detected heterogeneous bacteria and a high incidence of streptococci compared with that observed using cultivation methods. In addition, the results of our study may indicate a lower incidence of MRSA than previously expected in HCAP patients.     

Comparison of conventional and molecular methods for the detection of bacterial pathogens in sputum samples from cystic fibrosis patients

The nature of the micro-flora present in sputa of six different cystic fibrosis (CF) patients was assessed using routine microbiological culture and molecular methods. Bacterial genes for the small subunit ribosomal RNA (ssu rDNA) were specifically amplified from DNA extracted from the sputum samples, cloned and characterised by hybridisation and DNA sequencing. A large number of clones from six sputa were screened. Initially, oligonucleotide hybridisation was performed with five probes, specific for Gram-positives and Gram-negatives in general and the main pathogens for the CF patient (Staphylococcus aureus, Pseudomonas aeruginosa and Haemophilus influenzae). For a single sputum sample, the results were fully congruent when culture and molecular methods were compared. In the other five sputa, discrepancies for S. aureus and/or H. influenzae were documented. Although S. aureus DNA and H. influenzae DNA was detected in three and four sputa, respectively, strains could not be cultured. Although the PCR approach is not capable of distinguishing viable from dead bacteria, all of the CF patients had a history of S. aureus infections, while one of the CF patients once had cultivable H. influenzae in the sputum as well. A number of clones for probe-unidentified Gram-negative or Gram-positive bacterial species were further analysed by sequencing and additional potential pathogens were identified. Although routine culture of sputum frequently points to mono-specific exacerbations, our molecular data indicate that the other CF-related pathogens appear to be persistently present as well. We conclude that routine culture for bacterial pathogens from CF sputa yields limited microbiological information since it frequently fails to identify a number of pathogenic bacterial species that are potentially present in a viable status in the lungs of these patients.     

Real-Time Fluorescence Loop Mediated Isothermal Amplification for the Detection of Acinetobacter baumannii

Background

Detection of Acinetobacter baumannii has been relying primarily on bacterial culture that often fails to return useful results in time. Although DNA-based assays are more sensitive than bacterial culture in detecting the pathogen, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. In addition, these molecular tools require expensive laboratory instruments. Therefore, establishing molecular tools for field use require simpler molecular platforms. The loop-mediated isothermal amplification method is relatively simple and can be improved for better use in a routine clinical bacteriology laboratory. A simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in the same platform has been developed in recent years. This method is referred to as real-time loop-mediated isothermal amplification. In this study, we attempted to utilize this method for rapid detection of A. baumannii.

Methodology and Significant Findings

Species-specific primers were designed to test the utility of this method. Clinical samples of A. baumannii were used to determine the sensitivity and specificity of this system compared to bacterial culture and a polymerase chain reaction method. All positive samples isolated from sputum were confirmed to be the species of Acinetobacter by 16S rRNA gene sequencing. The RealAmp method was found to be simpler and allowed real-time detection of DNA amplification, and could distinguish A. baumannii from Acinetobacter calcoaceticus and Acinetobacter genomic species 3. DNA was extracted by simple boiling method. Compared to bacterial culture, the sensitivity and specificity of RealAmp in detecting A. baumannii was 98.9% and 75.0%, respectively.

Conclusion

The RealAmp assay only requires a single unit, and the assay positivity can be verified by visual inspection. Therefore, this assay has great potential of field use as a molecular tool for detection of A. baumannii.     

Diagnosis methods for pneumococcal etiology determination in lower respiratory tract infections

A comparative study of etiological diagnosis in lower respiratory tract infections (LRTI), by conventional bacteriological methods and by pneumococcal antigen direct detection in sputum was performed. This work followed the establishing of rapid methods place, respectively of coagglutination (CoA), counterimmunoelectrophoresis (CIE), within the methodology of bacteriological diagnosis in lower respiratory tract infections presenting pneumococcal etiology. The results of investigations performed on 84 sputa from LRTI patients proved the utility of CoA method in determining a rapid etiological diagnosis, important for applying an emergence targetted antibiotherapy. CoA method, with the reagents in use, covering only 10 out of 83 serological types of S. pneumoniae in not capable of replacing conventional methods of bacteriological diagnosis; they complete each other, increasing the efficiency of etiological diagnosis in LRTI. CIE method is less sensitive and more difficult to perform, being less useful in rapid etiological diagnosis of LRTI.     

痰标本涂片检查与培养结果分析

目的：探讨痰标本涂片检查在细菌培养以及临床治疗中的意义。方法：送检标本首先直接涂片革兰染色镜检,同时将合格痰标本划分为以阴性杆菌、阳性球菌、霉菌为主和普通标本四大类。再对合格痰标本行细菌学培养及药物敏感测定。结果：314份痰标本合格率为62．4％(196／314)。196份合格痰标本共检出145株病原菌,培养阳性率74．0％。涂片以阴性杆菌、阳性球菌、霉菌为主的标本与培养结果符合率分别为93．1％、68．4％和41．2％。结论：痰培养结果的准确性受痰标本是否合格、病原菌的生长速度以及是否使用过大剂量抗生素等因素直接影响。临床医生必须依据临床症状及涂片结果判断作出何种为真正的病原菌。     

呼吸道感染病原菌分析

目的 了解老年人院内呼吸道感染与非老年人呼吸道感染病原菌分布情况。方法 回顾性调查１９９８年１月至１２月老年人院内呼吸道感染（老年组）的２８５份痰标本和同时期非老年人呼吸道感染（非老年组,含门诊和住院）的１１９２份痰标本的培养情况。结果 老年组阳性率为８９．１％（２５４／２８５）,其中２２份标本为混合感染,共检出病原菌２７６株,以非发酵菌占绝对优势（９７．８％）;非老年组阳性率为４０．３％（４８０／１１９２）,病原菌无明显集中趋势,非发酵菌２８．３％,肠杆菌科２３．８％,革兰阳性菌２５．０％,真菌２２．９％。结论 老年组阳性率明显高于非老年组（Ｘ＾２＝２１９．６,Ｐ〈０．００５）,病原菌以非发酵菌为主,非老年组病原菌各科细菌所占比例无明显差异,引起呼吸道感染的病原菌主要为条件致病菌。     

Bronchoscopic Diagnosis of Pulmonary Infections—Comparison of Protected-Specimen Brush and Cytology Brush With Lung Aspirates

In a recent study the use of a new plugged double-lumen protected-specimen brush with the flexible fiberoptic bronchoscope was advocated to isolate pathogens in lower respiratory tract infections while avoiding upper respiratory tract contamination. To compare the efficacy of this brush and a standard single-lumen cytology brush in identifying the etiologic agent in lower respiratory tract infections, we studied 18 patients with lung infections. Transthoracic lung aspiration was done in all but two patients in an attempt to identify the specific etiologic agent. In these two cases, cultures of specimens of blood or postmortem lung tissue yielded the causative organism. In 12 patients anaerobic or aerobic bacteria (or both) were identified, whereas one patient had a mixed bacterial and fungal infection. Using the cytology brush and the protected-specimen brush we identified at least one pathogen in 10 of 12 and 10 of 13 cases, whereas both brushes missed one or more causative organisms in 8 of 12 and 8 of 13 cases, respectively. Nonetiologic organisms were found in 8 of 12 cases by the cytology brush and 8 of 13 cases by the protected-specimen brush. Quantitative culture techniques improved the specificity of the brush results in infections where aerobes predominated. Our data show that bronchoscopic cultures of lower respiratory tract infections do not consistently recover the causative agent and are frequently subject to contamination by nonetiologic organisms. There was no difference between the brushes in avoiding contamination.     

Cytopathologic and genetic diagnosis of pulmonary amebiasis: a case report

BACKGROUND: Amebiasis is a parasitic infection with Entamoeba histolytica. Pulmonary amebiasis is rare since the infection is commonly manifested as amebic colitis or liver abscess. Most pleuropulmonary amebiasis is seen in patients with amebic liver abscesses. A pulmonary amebic lesion without either a liver abscess or amebic colitis is extremely rare. Thus, reported cases of sputum cytologic diagnosis of a pulmonary amebic lesion from a patient without a liver abscess are also very rare. CASE: A 53-year-old man presented with a dry cough and mild fever. Chest radiography revealed an abnormal solitary mass lesion in the right upper lung field. The clinical diagnosis was a bacterial lung abscess. Sputum cytologic examination demonstrated many trophozoites of E. histolytica. Following sputum cytodiagnosis, serologic tests revealed a slightly high but almost normal titer of IgG antibodies to E. histolytica, indicating the possible presence of the pathogen. Polymerase chain reaction (PCR) using E. histolytica-specific primers for DNA extracted from the sputum sample revealed specific DNA product. CONCLUSION: Pulmonary amebiasis without either a liver abscess or amebic colitis must be distinguished from bacterial abscesses and neoplastic disease. A sputum cytologic examination combined with PCR for DNA extracted from a sputum sample is a good approach to the diagnosis of a pulmonary amebic abscess.     

Profiling of the bacteria responsible for pyogenic liver abscess by 16S rRNA gene pyrosequencing

Pyogenic liver abscess (PLA) is a severe disease with considerable mortality and is often polymicrobial. Understanding the pathogens that cause PLA is the basis for PLA treatment. Here, we profiled the bacterial composition in PLA fluid by pyrosequencing the 16S ribosomal RNA (rRNA) gene based on next-generation sequencing (NGS) technology to identify etiological agents of PLA and to provide information of their 16S rRNA sequences for application to DNA-based techniques in the hospital. Twenty patients with PLA who underwent percutaneous catheter drainage, abscess culture, and blood culture for isolates were included. Genomic DNAs from abscess fluids were subjected to polymerase chain reaction and pyrosequencing of the 16S rRNA gene with a 454 GS Junior System. The abscess and blood cultures were positive in nine (45%) and four (20%) patients, respectively. Pyrosequencing of 16S rRNA gene showed that 90% of the PLA fluid samples contained single or multiple genera of known bacteria such as Klebsiella, Fusobacterium, Streptococcus, Bacteroides, Prevotella, Peptostreptococcus, unassigned Enterobacteriaceae, and Dialister. Klebsiella was predominantly found in the PLA fluid samples. All samples that carried unassigned bacteria had 26.8% reads on average. We demonstrated that the occurrence of PLA was associated with eight known bacterial genera as well as unassigned bacteria and that 16S rRNA gene sequencing was more useful than conventional culture methods for accurate identification of bacterial pathogens from PLA.     

Spindle cell conversion by Kaposi's sarcoma-associated herpesvirus: formation of colonies and plaques with mixed lytic and latent gene expression in infected primary dermal microvascular endothelial cell cultures

Angiogenic Kaposi's sarcoma (KS) skin lesions found in both AIDS and non-AIDS patients are universally associated with infection by the presumed causative agent, known as KS-associated herpesvirus (KSHV) or human herpesvirus 8. KSHV genomes expressing latent state virus-encoded mRNAs and the LANA1 (latent nuclear antigen 1) protein are consistently present in spindle-like tumor cells that are thought to be of endothelial cell origin. Although the KSHV lytic cycle can be induced in rare latently infected primary effusion lymphoma (PEL) cell lines, the ability to transmit or assay infectious KSHV has so far eluded investigators. Here, we demonstrate that infection with supernatant virions derived from three different tetradecanoyl phorbol acetate-induced PEL cell lines can induce cultured primary human dermal microvascular endothelial cells (DMVEC) to form colonies of proliferating latently infected spindle-shaped cells, all of which express the KSHV-encoded LANA1 protein. Although their initial infectivity varied widely (JSC1 > > BC3 > BCP1), virions from all three cell lines produced distinctive spindle cell colonies and plaques without affecting the contact-inhibited cobblestone-like phenotype of adjacent uninfected DMVEC. Each infected culture could also be expanded into a completely spindloid persistently infected culture displaying aggregated swirls of spindle cells resembling those in KS lesions. Formation of new colonies and plaques was inhibited in the presence of phosphonoacetic acid or gangciclovir, but these antiherpesvirus agents had little effect on the propagation of already latently infected spindloid cultures. In persistently infected secondary cultures, patches of up to 10% of the spindloid cells constitutively expressed several early viral lytic cycle proteins, and 1 to 2% of the cells also formed typical herpesvirus DNA replication compartments, displayed cytopathic rounding effects, and expressed late viral antigens. We conclude that de novo KSHV infection induces a spindle cell conversion phenotype in primary DMVEC cultures that is directly associated with latent state expression of the LANA1 protein. However, these cultures also spontaneously reactivate to produce an unusual combination of both latent and productive but slow lytic cycle infection. The formation of spindle cell colonies and plaques in DMVEC cultures provides for the first time a quantitative assay for directly measuring the infectivity of KSHV virion preparations.     

Virus contaminations of cell cultures – A biotechnological view

In contrast to contamination by microbes and mycoplasma, which can be relatively easily detected, viral contamination present a serious threat because of the difficulty in detecting some viruses and the lack of effective methods of treating infected cell cultures. While some viruses are capable of causing morphological changes to infected cells (e.g. cytopathic effect) which are detectable by microscopy some viral contaminations result in the integration of the viral genome as provirus, this causes no visual evidence, by means of modification of the cellular morphology. Virus production from such cell lines, are potentially dangerous for other cell cultures (in research labs)by cross contaminations, or for operators and patients (in the case of the production of injectable biologicals) because of potential infection. The only way to keep cell cultures for research, development, and the biotech industry virus-free is the prevention of such contaminations. Cell cultures can become contaminated by the following means: firstly, they may already be contaminated as primary cultures (because the source of the cells was already infected), secondly, they were contaminated due to the use of contaminated raw materials, or thirdly, they were contaminated via an animal passage. This overview describes the problems and risks associated with viral contaminations in animal cell culture, describes the origins of these contaminations as well as the most important virsuses associated with viral contaminations in cell culture. In addition, ways to prevent viral contaminations as well as measures undertaken to avoid and assess risks for viral contaminations as performed in the biotech industry are briefly described. This revised version was published online in August 2006 with corrections to the Cover Date.     

Specific detection and confirmation of Campylobacter jejuni by DNA hybridization and PCR.

Conventional detection and confirmation methods for Campylobacter jejuni are lengthy and tedious. A rapid hybridization protocol in which a 1,475-bp chromogen-labelled DNA probe (pDT1720) and Campylobacter strains filtered and grown on 0.22-micron-pore-size hydrophobic grid membrane filters (HGMFs) are used was developed. Among the environmental and clinical isolates of C. jejuni, Campylobacter coli, Campylobacter jejuni subsp. doylei, Campylobacter lari, and Arcobacter nitrofigilis and a panel of 310 unrelated bacterial strains tested, only C. jejuni and C. jejuni subsp. doylei isolates hybridized with the probe under stringent conditions. The specificity of the probe was confirmed when the protocol was applied to spiked skim milk and chicken rinse samples. Based on the nucleotide sequence of pDT1720, a pair of oligonucleotide primers was designed for PCR amplification of DNA from Campylobacter spp. and other food pathogens grown overnight in selective Mueller-Hinton broth with cefoperazone and growth supplements. All C. jejuni strains tested, including DNase-producing strains and C. jejuni subsp. doylei, produced a specific 402-bp amplicon, as confirmed by restriction and Southern blot analysis. The detection range of the assay was as low as 3 CFU per PCR to as high as 10(5) CFU per PCR for pure cultures. Overnight enrichment of chicken rinse samples spiked initially with as little as approximately 10 CFU/ml produced amplicons after the PCR. No amplicon was detected with any of the other bacterial strains tested or from the chicken background microflora. Since C. jejuni is responsible for 99% of Campylobacter contamination in poultry, PCR and HGMF hybridization were performed on naturally contaminated chicken rinse samples, and the results were compared with the results of conventional cultural isolation on Preston agar. All samples confirmed to be culture positive for C. jejuni were also identified by DNA hybridization and PCR amplification, thus confirming that these DNA-based technologies are suitable alternatives to time-consuming conventional detection methods. DNA hybridization, besides being sensitive, also has the potential to be used in direct enumeration of C. jejuni organisms in chicken samples.     

Microbial hazards in plant tissue and cell cultures

Summary A wide range of microorganisms (filamentous fungi, yeasts, bacteria, viruses and viroids) and micro-arthropods (mites and thrips) have been identified as contaminants in plant tissue cultures. Contaminant may be introduced with the explant, during manipulations in the laboratory or by micro-arthropod vectors. Contaminants may express themselves immediately or can remain latent for long periods of time. This often makes it difficult to identify the source of contamination. Disinfection protocols have now been developed for a wide range of plant species including those infected with viruses/viroids or endophytic bacteria. They may include the selection of pathogen-free donor plants or donor plant treatments such as thermotherapy. Also microbiological quality assurance systems (e.g. Hazard Analysis Critical Control Point; HACCP procedures) have been adapted to the needs of commercial plant tissue culture laboratories. These are aimed at, preventing the introduction of pathogens, into tissue cultures at establishment and in the laboratory. In established in vitro cultures preventative strategies have proved to be essential, since it is extremely difficult to eliminate environmental bacterial and fungal contaminants using, antibiotics and fungicides. In many cases anti-microbial treatments only inhibit contaminants and low levels of contamination persist. In particular, the use of antibiotics against Gram-negative bacteria (including plant pathogenic bacteria and Agrobacterium tumefaciens vector systems used in genetic engineering) has been shown frequently to be extremely difficult or unsuccessful. Detection of latent contamination may involve the use of general and semi-selective microbial growth media or serological and PCR-based molecular techniques for specific pathogens. However, it is often difficult to detect low numbers of latent bacterial contaminants (e.g. levels present following antibiotic treatment or when acidified plant media are used). This poses a particular risk in the production of transgenic plants where the elimination or detection of Agrobacterium tumefaciens-based vector systems cannot be guaranteed with the currently available methodologies. Recent research has also shown that there is a risk of the transmission of human pathogens in plant tissue cultures.     

ICU医院内深部真菌感染临床分析

目的研究ICU深部真菌感染的临床及病原学特点。方法收集129例ICU深部真菌患者的血、痰、粪便、尿、腹腔引流液培养结果及其临床资料。结果129例ICU深部真菌患者中共分离出152株真菌,以白色念珠菌及热带念珠菌为主,其中白色念珠菌占55．92％,热带念珠菌占21．71％。感染部位以尿路感染最常见,占50．66％,其次为呼吸道感染,占29．61％。结论ICU深部真菌感染菌种和部位有自己的特点,并且有一定的诱发因素,应予以高度重视。     

Netilmicin in the treatment of bacterial respiratory tract infections

Netilmicin - a semisynthetic aminoglycoside - was administered to 33 patients with the acute or chronic lower respiratory tract or pulmonary infections in a daily dose of 5 mg/kg body weight for 10 days. A principle criterium of patients classification to netilmicin therapy were sensitive bacterial strains either in sputum or in BAL liquid. A significant clinical improvement was noted in 88% of the treated patients. However, elimination of pathogens from the sputum was achieved only in 52% of these patients. No improvement was observed in 4% of the treated patients. No adverse reactions were noted. Netilmicin proved safe and effective antibacterial agent in patients with respiratory infections.