Tuberculosis in Indonesia: recent studies on detection and drug susceptibility of mycobacteria in Jakarta.

Sputum specimens from more than 1000 Indonesian tuberculosis suspects were examined by bacteriologic culture, and by bright field and fluorescence microscopy. Two hundred twenty had positive Mycobacterium tuberculosis cultures and of these 68% were positive by fluorescence microscopy. Agreement between culture, both negative and positive, and fluorescence microscopy was 87%. Sensitivity to antituberculous drugs was performed in 209 isolates. Significant resistance to isoniazid, para-amino salicylic acid, and streptomycin was found i.e. 65 (31%), 19 (19%), 54 (26%), respectively. Fluorescence microscopy was a useful method for rapid microscopic confirmation of tuberculosis and was especially valuable in detecting difficult-to-culture organisms.     

Genotyping of potentially pathogenic Acanthamoeba strains isolated from nasal swabs of healthy individuals in Peru

Free Living Amoebae (FLA) of Acanthamoeba genus are widely distributed in the environment and can be found in the air, soil and water; and have also been isolated from air-conditioning units. In humans, they are causative agents of a sight-threating infection of the cornea, Acanthamoeba keratitis (AK) and a fatal infection of the central nervous system known as Granulomatous Amoebic Encephalitis (GAE). In this study, a survey was conducted in order to determine the presence and pathogenic potential of free-living amoebae of Acanthamoeba genus in nasal swabs from individuals in two regions of Peru. Identification of isolates was based on cyst morphology and PCR-sequencing of the Diagnostic Fragment 3 to identify strains at the genotype level. The pathogenic potential of the isolates was also assayed using temperature and osmotolerance assays and extracellular proteases zymograms. The obtained results revealed that all isolated strains exhibited pathogenic potential. After sequencing the highly variable DF3 (Diagnostic Fragment 3) region in the 18S rRNA gene as previously described, genotype T4 was found to be the most common one in the samples included in this study but also genotype T15 was identified. To the best of our knowledge, this is the first study on the characterization of Acanthamoeba strains at the genotype level and the first report of genotype T4 and T15 in Peru.     

Acanthamoeba T3, T4 and T5 in swimming-pool waters from Southern Brazil

Species of Acanthamoeba, known to cause keratitis (AK) and granulomatous encephalitis in humans are frequently isolated from a variety of water sources. In this study, 13 Acanthamoeba isolates from swimming pools were classi?ed at the genotype level based on the sequence analysis of the Acanthamoeba small-subunit rRNA gene. Nine of the 13 isolates were genotype T5, three were genotype T4, and one was T3. Several genotypes have been reported worldwide as causative agents of AK, including genotypes T3, T4, and T5. The present study indicates that genotype T5 is a common contaminant in swimming-pool water.     

抗酸染色全自动显微扫描识别系统的临床应用评价

目的 评价抗酸染色全自动显微扫描系统(AFB-AS)检测肺结核患者痰液标本应用价值。方法 搜集2019年1月至8月于北京结核病控制研究所确诊的462例肺结核患者作为研究对象,每例患者留取3份初诊痰液标本,共计1386份。所有标本均完成萋-尼抗酸染色,采用双盲法使用传统抗酸杆菌染色镜检和AFB-AS检查;每例患者选取2份痰液标本进行分枝杆菌分离培养,共计完成924份;其中1份同时进行GeneXpert MTB/RIF(简称“Xpert”)检测,共计完成462份。比较不同方法检测肺结核患者痰标本结果;以分枝杆菌分离培养结果为参照,分析传统抗酸杆菌染色镜检和AFB-AS检测肺结核患者痰标本的效能;分析传统抗酸杆菌染色镜检和AFB-AS检测均为阳性结果的等级相关性。结果 各方法检测肺结核患者痰标本阳性率从高到低依次为Xpert(69.26%,320/462)、分枝杆菌分离培养(43.61%,403/924)、AFB-AS(43.22%,599/1386)、传统抗酸杆菌染色镜检(38.24%,530/1386)。AFB-AS检测痰标本阳性率明显高于传统抗酸杆菌染色镜检,差异有统计学意义(χ²=67.015,P<0.01)。AFB-AS与传统抗酸杆菌染色镜检判读阳性结果等级的相关系数为0.954。以分枝杆菌分离培养结果为参照,AFB-AS检查肺结核患者痰标本敏感度为94.29%(380/403)、特异度为90.40%(471/521)、阳性预测值为88.37%(380/430)、阴性预测值为95.34%(471/494),Kappa值为0.841。结论 AFB-AS检测肺结核患者痰液标本具有较好的应用价值,检验性能能够满足实施萋-尼抗酸染色查找抗酸杆菌项目的要求。     

不同细菌学与病理学检查技术对骨关节结核诊断的效能研究

目的:回顾性分析脓液标本细菌学检测技术和组织标本病理学检测技术诊断骨关节结核的检测效能。方法:以2016年1月至2018年12月在北京胸科医院住院治疗的213例送检同部位脓液和组织标本进行检测的疑似骨关节结核患者作为研究对象,取新鲜脓液标本进行涂片染色镜检、GeneXpert MTB/RIF(GeneXpert)检测、实时荧光定量PCR(FQ-PCR)检测和BACTEC MGIT 960液体培养(MGIT 960液体培养);组织标本经石蜡包埋后进行抗酸染色镜检和FQ-PCR检测。结果:根据骨结核临床诊断综合参考标准(CRS),186例纳入患者诊断为骨关节结核,其中124例为确诊结核患者,23例为高度疑似结核患者,39例为疑似结核患者;27例患者排除骨关节结核。与CRS比较,脓液标本涂片镜检、MGIT 960液体培养、FQ-PCR、GeneXpert以及组织标本抗酸染色和FQ-PCR检测的敏感度分别为28.7%(49/171)、49.0%(48/98)、58.1%(25/43)、87.5%(161/184)、48.6%(90/185)和85.4%(146/171);特异度分别为100.0...     

贾第虫纯培养的建立

从腹泻患儿新鲜粪便内,浓集、纯化贾第虫包囊。用生理盐水制成悬液,感染长爪沙鼠(Meriones unguiculatus)乳鼠。接种后第8d将受染鼠处死。用无菌术从上段小肠分离滋养体,接种于改良TYI-S-33培养基内,于37℃培养。培养后第14d,生长旺盛的虫体在培养管壁内面形成密集的细胞单层。生长高峰期和倍增时间分别在培养后的第120和15±2h。各批虫体经液氮冷冻复苏后,虫体复活率为53～80％。复苏虫体生长良好。本纯培养已维持9月余,传代100多次。     

CDG-3荧光探针检测结核分枝杆菌的初步研究

目的 探讨CDG-3荧光探针[β-内酰胺酶(BlaC)特异性绿色荧光探针]用于结核病诊断的潜在可能性。方法 为明确CDG-3荧光探针的特异性,选取结核分枝杆菌(Mycobacterium tuberculosis,MTB)标准株H37Rv,牛和非洲分枝杆菌标准株,35种临床相对常见的不同非结核分枝杆菌(nontuberculous mycobacteria,NTM)标准株和6种呼吸道感染病原菌标准株,与CDG-3荧光探针反应后进行检测;为初步了解CDG-3荧光探针临床应用效果,收集2016年1月至9月于北京胸科医院门诊就诊的200例疑似肺结核患者的痰标本分别进行金胺O荧光染色法、培养法(罗氏固体培养联合MGIT960液体培养,二者只要有一种结果为阳性即认为培养阳性)和CDG-3荧光探针检测,并对检测结果进行分析。结果 以H37Rv为标准,CDG-3荧光探针检测牛和非洲分枝杆菌标准株结果均为阳性。试验的35种NTM标准株中,CDG-3荧光探针检测结果阳性的菌株仅有3株,分别是堪萨斯分枝杆菌、玛尔摩分枝杆菌和草分枝杆菌;其余32株的检测结果为阴性。6种呼吸道感染病原菌中,5种检测结果为阴性,1种(诺卡菌)为阳性。在临床痰标本实验中,CDG-3荧光探针对痰MTB的阳性检出率为53.0%(106/200),高于金胺O荧光染色法(29.5%,59/200)与培养法(35.0%,70/200)的阳性率,差异有统计学意义(χ ²值分别为33.587、21.121,P值均为0.000)。以培养法结果为标准,CDG-3荧光探针检测的敏感度为84.3%(59/70),高于金胺O荧光染色法(70.0%,49/70);特异度为63.8%(83/130),低于金胺O荧光染色法(92.3%,120/130);以临床诊断为标准,CDG-3荧光探针检测的敏感度为64.4%(96/149),高于金胺O荧光染色法(39.6%,59/149)和培养法(44.3%,66/149);特异度为95%(19/20),低于金胺O荧光染色法(100%,20/20),高于培养法(90%,18/20)。 结论 CDG-3荧光探针对MTB的选择性较好,受NTM干扰较小,与呼吸道感染病原菌的反应还需进一步研究;CDG-3荧光探针技术具有快速、简便、检出效率高等优势,但特异度有待进一步提高。     

实时荧光PCR技术在不同类型结核病诊断中的应用价值

目的 分析比较实时荧光PCR与涂片镜检、培养三种方法在结核杆菌检测的差异.方法 对我院疑似结核病患者的临床标本同时进行实时荧光PCR检测、涂片镜检和培养,将三组检测结果进行比较.结果 215例疑似肺结核患者痰标本同时采用实时荧光PCR、涂片镜检和培养三种方法检测,阳性检出率分别为46.5％、32.6％和44.7％.实时荧光PCR法与涂片镜检法的阳性率间比较有统计学意义,实时荧光PCR法与培养法的阳性率同比较无统计学意义.112例疑似其他类型结核病患者的胸腹水、脑脊液、穿刺液和尿液等非痰标本同时采用上述三种方法检测,阳性率分别为18.8％、3.6％和16.1％.统计学意义同上.结论 实时荧光PCR检测技术在临床上对结核病的诊断和鉴别具有较好的应用价值.     

Prevalence of Trichomonas vaginalis in some Filipino women

Vaginal specimens obtained from 1,284 hospitality girls and 87 expectant mothers were examined for Trichomonas vaginalis by first examining material collected from vaginal swabs and after incubation in Feinberg and Whittington culture medium. Twenty-four percent of the specimens examined, shortly after the cotton-tip swab was placed into 1 ml culture medium, were positive by direct microscopic examination and 37% positive following 3 to 5 days incubation at 37 degrees C. Only 3 of the specimens from mothers were positive after the first examination and 4 positive after culture. These high prevalence rates are expected among hospitality girls and are the highest rates thus far reported from the Philippines. These results provide convincing evidence of the value of using cultural methods in determining prevalence rates for Trichomonas vaginalis infections in females.     

A multicenter study of a new Helicobacter pylori selective medium. Columbia horse blood agar HP

We conducted a study for the growth of and selectivity for the desired microorganisms using a newly developed selective culture medium for Helicobacter pylori, Columbia horse blood agar HP (CHBHP), at three different Japanese clinical laboratories, Hokkaido, Kanto and Kyusyu. When standard strains and clinical isolates of H. pylori were examined, the recovery of the organism on the CHBHP media was comparable to that of conventional selective and nonselective media. However, colonies were obviously larger on the CHBHP media. These media yielded the highest H. pylori positive rate for clinical specimens at all the three laboratories. The detection rate of the CHBHP media in H. pylori-positive specimens was higher than that of media commonly used at the three laboratories (98.1% to 100% vs. 88.0% to 96.2%). The CHBHP media also achieved a higher detection rate for specimens from H. pylori-infected animals. CHBHP media have an excellent growth supporting ability and selectivity originating from Columbia agar base and do not require the combined use of non-selective media for the growth and isolation of the organism, resulting in lower cost. Thus, they are useful media for the selective culture and isolation of H. pylori from clinical and animal specimens.     

Experimental keratitis in rats caused by Acanthamoeba griffini: A kinetic histopathological study

The aim of this study was to evaluate the inflammation process that resulted from the inoculation of Wistar Rats with Acanthamoeba griffini, a virulent T3 Acanthamoeba genotype that produces keratitis. Haematoxylin and eosin, periodic acid stain, immunohistochemistry and morphometry were used to analyse tissues from rats of an Acanthamoeba keratitis (AK) model. Two weeks after inoculating the rats with A griffini trophozoites, the thickness of the stroma had diminished, followed by an increase in thickness at 4 weeks. At the latter time, an abundance of inflammatory infiltrate cells was observed, some found to express IL-1β, IL-10 and/or caspase 3. Intercellular adhesion molecule-1 was expressed in corneal blood vessels amid the abundant vascularization characteristic of the development of AK. Through an immunohistochemical technique, trophozoites were detected at 2 and 4 weeks post-inoculation. By 8 weeks, there were a low number of trophozoites and cysts and the corneas of infected rats were similar in thickness to those of the controls. Thus, the rats were capable of healing experimental AK in the present rat model. Diverse immunological mechanisms regulated the inflammatory process in acute AK induced by A griffini in a murine model.     

Comparison of three methods for decontamination of faeces for isolation of Mycobacterium tuberculosis.

Sputum and faeces were obtained from 276 patients on admission to a study of drug resistance in Hong Kong. Acid-fast bacilli were detected microscopically in 103 (37%) sputum specimens and 135 (49%) yielded Mycobacterium tuberculosis on culture. Three methods were used to decontaminate faeces prior to dilution and culture in selective liquid Kirchner medium. A total of 61 faecal specimens were positive for M. tuberculosis on culture and, of these, pretreatment with sodium hydroxide yielded 60 (98%), Portaels modification of Wolinsky and Rynearsons's method 28 (46%) and the combined use of benzalkonium chloride and 1-hexdecylpyridinium chloride yielded 32 (52%). It is recommended that faeces should be treated with sodium hydroxide followed by dilution and culture in selective media, although it may be necessary to formulate new selective media for mycobacterial species other than M. tuberculosis.     

几种培养幽门螺杆菌方法的比较

背景：全球50％以上的人口感染幽门螺杆菌（H.pylori）,对其培养方法的研究非常重要。目的：研究不同培养基对H.pylori生长的影响。方法：制备固体和液体培养基,并加入脱纤维绵羊血和3种动物血清（羊、马、牛）行且pytori培养。以细菌形态、涂片镜检、快速尿素酶试验、PCR法和菌体浓度鉴定H.pylori。结果：添加脱纤维绵羊血和动物血清的固体和液体培养基均可成功培养出且pylori,其中脱纤维绵羊血的固体培养基扩菌浓度最高．添加马血清的固体和液体培养基的H．pylori扩菌效果均较理想。结论：固体和液体培养基均可取得较为满意的H.pylori扩菌效果．其中添加马血清的培养基更易于分装和保存．值得推广．     

Improved recovery of Mycobacterium tuberculosis from pleural aspirates: bedside inoculation, heparinized containers and liquid culture media

The effects of bedside inoculation, heparinized containers and liquid culture media on the recovery of Mycobacterium tuberculosis from pleural aspirates were evaluated in this study. Of 155 patients, 63 were diagnosed to have pleural effusion tuberculous in origin. The overall recovery of M. tuberculosis was 57.1%. Bedside inoculation of the specimens produced a significantly higher yield than laboratory inoculation using non-heparinized specimens. When the pleural aspirates were transported in heparinized containers, the recovery rate was comparable to that from bedside inoculation, but lower when non-heparinized containers were used. No significant difference was found in recovery rate between the two liquid media, but the rate was significantly higher with the use of liquid media than conventional solid media. Thus, bedside inoculation of pleural aspirates, use of heparinized containers for transport for delayed inoculation in the laboratory and use of liquid culture media are recommended.     

Laboratory diagnosis of systemic fungal diseases

The increase in the number of fungal infections seen in debilitated and immunocompromised patients in the last several years makes it necessary to consider all fungi as potential pathogens. Clinical microbiology laboratories are playing increasingly important roles in the recovery, isolation, and identification of these fungi. This article contains specific recommendations and references concerning a practical approach to the laboratory identification of systemic fungi. The proper and timely selection, collection, and transport of specimens is imperative, and clinicians are responsible for appropriate specimen selection to ensure optimal chances of recovery of pathogens. Respiratory tract secretions and blood are excellent sources for detection of disseminated fungal infection. Specimens should be placed into transport media if the sample size is small or if only a small number of organisms are thought to be present. Direct microscopic examination of specimens can provide valuable information, often allowing a clinician to initiate immediate therapy. Specimens that are more likely than others to contain systemic fungi and that should be examined routinely include the following: pulmonary biopsy material, bronchial washes and lavages, specimens from immunocompromised patients, purulent specimens, and specimens suspected of containing a specific fungus. Valuable methods of examining specimens directly include treatment with KOH and calcofluor white. Use of media to recover fungi is the basis of making a laboratory diagnosis of a fungal disease, and the use of proper recovery and subculture media is imperative. Noninhibitory media allow contaminants to grow readily and should be used only to recover fungi from normally sterile body sites or for subculture. Blood-enriched media allow almost all pathogenic and saprophytic fungi to flourish. Therefore, such media, unless they contain antibiotics, should not be used as primary recovery media. Media that contain antibiotics should be used as primary recovery media to prevent overgrowth of pathogenic fungi by contaminants. Yeasts recovered from clinical specimens can be identified by a combination of tests, which include direct microscopic examination, germ tube formation, microscopic morphology of growth on corn meal agar, and ability to utilize certain carbohydrates. Molds recovered from clinical specimens are identified by a combination of growth rate, colonial characteristics, size and shape of hyphae, and microscopic examination of reproductive structures and other fungal elements.(ABSTRACT TRUNCATED AT 400 WORDS)     

幽门螺杆菌粪便抗原检测——一种新的非侵入性诊断方法

目的：评估幽门螺杆菌粪便抗原(HpSA）榆测诊断Hp感染的可靠性。方法：收集43例接受胃镜检查患者的粪便标本,用一种市售的ELISA试剂盒检测HpSA：以尿素酶试验．培养和涂片染色检测Hp作为“金标准”,培养和／或涂片染色阳性定为Hp感染。结果：“金标准”诊断Hp阳性24例,阴性19例。“金标准”阳性的24例HpSA检测均阳性,阴性的19例HpSA检测18例阴性和1例阳性。HpSA试验的敏感性为100％(24／24),特异性为94．7％(18／19),总的诊断准确率为97．6％(42／43)。结论：HpSA试验是一简便、非侵入性、准确诊断Hp感染的新方法。     

Case of laboratory cross-contamination of Mycobacterium tuberculosis in the broth-based culture system

We experienced a case of laboratory cross-contamination of Mycobacterium tuberculosis on the broth based culture system. These false-positive cultures were confirmed by analysis of DNA fingerprinting, RFLP method, which showed the same pattern in three specimens with that of the first manipulated specimen in our laboratory on that day, out of 7 specimens examed. We found possible several process causing cross-contamination where mixture of the foreign body could occur in buffer or NALC-NaOH. False-positive cultures for Mycobacterium tuberculosis may lead to unnecessary, potentially toxic, costly treatment, and changing the treatment strategy. So we must critically interpret a single positive culture, especially by liquid media.     

Evaluation of the performance of a novel sputum processing ReaSLR methodology for culture of sputum samples in solid and liquid media in comparison with modified Petroff’s method

BackgroundEarly case detection by sputum smear microscopy is a crucial step in the control of pulmonary tuberculosis in high burden countries. Due to low sensitivity of this rapid and cost effective method, culture of Mycobacterium tuberculosis (MTB) is considered as the gold standard. Modified Petroff's method using 2%–4% sodium hydroxide (NaOH) and N-acetyl- l-cysteine (NALC) to digest and at the same time to decontaminate the specimen is widely used in developing countries prior to culture. This method is considered tedious and cumbersome. A novel ReaSLR (ReaMetrix's Sputum Liquefying Reagent) methodology has been proposed as a simple and low-cost method for sputum processing. This study was undertaken to evaluate the performance of the ReaSLR method of sputum processing prior to culture in comparison to the modified Petroff's method.MethodsEarly morning sputum samples, collected from suspected TB patients, were divided into two equal halves and processed by two different methods i.e modified Petroff's method and ReaSLR method. After processing with different methods, each sample was inoculated in Lowenstein Jensen (LJ) medium and Mycobacteria growth indicator tube (MGIT). Smears were also prepared from the samples processed with modified Petroff's method and graded according to Centers for Disease Control and Prevention (CDC) grading after microscopic examination. Culture results of both the methods were recorded and analysed using SPSS 20.0 version.ResultsOn comparing different methods of sputum processing for culture in solid and liquid media, the rate of contamination in both the media was significantly high with ReaSLR method as compared with modified Petroff's method. Also, the mean time-to-detection of MTB growth in LJ medium was significantly less with modified Petroff's method i.e 30.21 days as compared to ReaSLR method (34.23 days; p < 0.001). However, the mean time-to-detection of MTB growth in MGIT was similar with both the methods.ConclusionDue to the high contamination rate in solid and liquid culture media, ReaSLR method cannot be considered as an alternative to modified Petroff's method for sputum processing prior to culture. The detection of growth of MTB in LJ media was also earlier with modified Petroff's method than ReaSLR method.     

骨关节结核病灶结核菌L型状况的观察

目的　探讨骨关节结核病灶中 ,结核菌L型的存在状况。方法 样本均采自骨科住院病人的脓液、肉芽、干酪物等 ,经碱液处理后 ,分别进行直接涂片,并接种在罗氏培养基上、Bactec培养液及结核菌L型培养液中 ,以查找结核杆菌及结核菌L型。结果 在 6 0例中 ,查到结核菌L型 11例(18.3% ),涂片阳性 9例 (15 % ) ,罗氏培养阳性 3例 (5 % ) ,快速培养阳性 12例 (2 0 % )。结论 对骨关节结核病灶进行结核菌培养的同时,加做结核菌L型检查是十分必要的。     

犬贾第虫纯培养的建立

将取自一1岁自然感染犬贾第虫的雌性、德国牧羊犬的包囊利用蔗糖密度梯度离心-G1耐酸漏斗滤过法进行分离和纯化,经口感染给10日龄的沙鼠幼鼠,8d后无菌取其小肠上段,分离出滋养体,转入改良TYS-I-33培养基中进行培养,在驯化5个月之后,虫体逐渐适应了培养环境,在管壁上形成了细胞单层并进行传代,每隔48~72h传代1次,共传18代。将培养物置肉汤及血琼脂平板培养,未见细菌及霉菌污染,为纯培养。