Molecular Detection of Legionella Pneumophila, Mycoplasma Pneumoniae and Chlamydia Pneumoniae among Sudanese Patients with Acute Respiratory Infections in Khartoum State, Sudan (original) (raw)
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Acta Microbiologica et Immunologica Hungarica, 2012
Legionella pneumophila is an important etiological agent in both hospital and community acquired pneumonia. The sensitivity of culture for isolation of L. pneumophila from clinical specimens is low and time consuming. Similar problem also exists when the method of direct immunofluorescence is used. To detect this organism quantitatively from respiratory specimens, a Taq Man based real-time PCR targeting the mip sequence was developed. Both real-time PCR and culture methods were applied on 262 respiratory specimens from 262 ICU patients with pneumonia admitted to 5 different hospitals in Tehran. The results of real-time PCR were compared with those obtained by culture. Real-time PCR and culture found 12 and 4 specimens, respectively, as positive for L. pneumophila. Its technical specificity (100%) was checked against a panel of microorganisms consisting of both Gram-positive and Gram-negative bacteria. Our real-time PCR assay showed high sensitivity (100%) and specificity (96.9%) and...
Epidemiology and Infection, 2018
Legionella pneumophila genotyping is important for epidemiological investigation of nosocomial and community-acquired outbreaks of legionellosis. The prevalence of legionellosis in pneumonia patients in the West Bank was monitored for the first time, and the sequence types (STs) from respiratory samples were compared with STs of environmental samples from different wards of the hospital. Sputum (n = 121) and bronchoalveolar lavage (BAL) (n = 74) specimens were cultured for L. pneumophila; genomic DNA was tested by 16S rRNA polymerase chain reaction (PCR) amplification. Nested PCR sequence-based typing (NPSBT) was implemented on DNA of the respiratory and environmental PCR-positive samples. Only one respiratory specimen was positive for L. pneumophila by culture. BAL gave a higher percentage of L. pneumophila-positive samples, 35% (26/74) than sputum, 15% (18/ 121) by PCR. NPSBT revealed the following STs: ST 1 (29%, 7/24), ST 461 (21%, 5/24), ST 1037 (4%, 1/24) from respiratory samples, STs from environmental samples: ST 1 (28.5%, 4/14), ST 187 (21.4%, 3/14) and ST 2070, ST 461, ST 1482 (7.1%, 1/14) each. This study emphasises the advantage of PCR over culture for the detection of L. pneumophila in countries where antibiotics are indiscriminately used prior to hospital admission. ST 1 was the predominant ST in both respiratory and environmental samples.
2017
Legionellae are gram-negative bacteria, rod shaped, strictly aerobic and nutritionally fastidious. Legionella species are implicated in two clinical syndromes: Legionnaires’ Disease (LD), and Pontiac fever, which are collectively known as legionellosis. Among the 56 species and 70 serogroups of Legionella species, Legionella pneumophila is the major cause of sporadic and outbreak legionellosis (91.5%), and serogroup 1 is the predominant serotype (84.2%). Many studies have demonstrated that the main source for LD is the potable water systems in large buildings like hospitals and hotels. The contamination of hospitals' water systems with Legionella is high risk for patients with various diseases, especially immunocompromised and those who may stay hospitalized for long period of time. LD is acquired by inhalation of aerosols contaminated with Legionella spp. or less commonly by aspiration of contaminated drinking water. Previous work in the Microbiology Research Laboratory at AQU ...
Folia Microbiologica, 2000
Polymerase chain reaction (PCR) was used for detecting Legionella DNA in water, sputum, tracheal aspirate and bronchoalveolar lavage fluid. There is paucity of data on the use of PCR for detection of Legionella in serum and urine samples. In 82 patients admitted with community-acquired pneumonia, urinary PCR was used in addition to urinary antigen assay for Legionella pneumophila serogroup 1 and serological tests (indirect immunofluorescence and ELISA) in paired sera. PCR was positive in urine samples from 21 patients (26 %): in six of seven patients with acute legionellosis by CDC criteria, and 15 patients with negative urine antigen showing no fourfold rise in antibody titers in immunofluorescence test.
Investigation of Legionella pneumophila and other Legionella species in atypical pneumonia patients
Türk hijiyen ve deneysel biyoloji dergisi, 2022
Investigation of Legionella pneumophila and other Legionella species in atypical pneumonia patients Atipik pnömonili hastalarda Legionella pneumophila ve diğer Legionella türlerinin araştırılması Kerim PARLAK 1 (ID), Ayşegül GÖZALAN 2 (ID), Sibel AYDOĞAN 3 (ID), Adem KOYUNCU 4 (ID), Hatice Canan HASANOĞLU 5 (ID), Selin NAR ÖTGÜN 6 (ID), Ziya Cibali AÇIKGÖZ 7 (ID) ÖZET Amaç: Bu çalışmada atipik pnömoni tanısı alan 50 hastada kültür, üriner antijen testi ve moleküler yöntemler kullanarak Legionella türlerinin araştırılması amaçlanmıştır. Yöntem: Solunum yolu örneklerinden Legionella türlerinin izolasyonu için seçici olmayan BCYE-α (Oxoid, İngiltere) ve seçici BMPA (Oxoid, İngiltere) besiyerleri kullanılmıştır. İdrar örneklerinde L. pneumophila serogrup 1'e özgü bakteriyel antijenin varlığı Alere BinaxNOW Legionella Üriner Antijen Kart (Abbott, ABD) testi ile araştırılmıştır.Tüm solunum yolu örnekleri Duplicα RealTime Legionella pneumophila 23S rRNA spesifik bölgesini saptayan (Euroclone Diagnostica, İtalya) ticari kit ve iki laboratuvar yapımı PCR yöntemi ile test edilmiştir. Laboratuvar yapımı jel elektroforez PCR testinde Legionella spp. için 16S ribozomal RNA gen kısmi dizilerinden tasarlanan Leg primerleri ve L. pneumophila için Lmip (macrophage infectivity potentiator) genini hedefleyen primerler ABSTRACT Objective: The aim of this study is to investigate Legionella species in 50 patients with atypical pneumonia, using culture, urinary antigen test and molecular techniques. Methods: Non-selective BCYE-α media (Oxoid, England) and selective BMPA media (Oxoid, England) were used to isolate Legionella spp. from respiratory tract samples. The urinary samples of the patients were tested with the Alere BinaxNOW Legionella Urinary Antigen Card (Abbott, US) test to identify the presence of L. pneumophila serogroup 1 specific bacterial antigen. All respiratory tractsamples were tested with a commercial Duplicα RealTime Legionella pneumophila 23S rRNA specific region detection kit (Euroclone Diagnostica, Italy) and two home-made PCR methods. Home-made gel electrophoresis PCR tests were performed using Leg primers designed from 16S ribosomal RNA gene partial sequences for Legionella spp and primers targeting the Lmip (macrophage
The American Journal of Tropical Medicine and Hygiene
Atypical pathogens including Mycoplasma pneumoniae and Legionella pneumophila are increasingly recognized as important causes of community-acquired pneumonia (CAP). Mycoplasma pneumoniae accounts for 20-40% of all CAP and L. pneumophila is responsible for 3-15% of cases. The paucity of data from India in this regard prompted us to conduct this prospective multicentric analysis to detect the prevalence of M. pneumoniae and L. pneumophila in our geographical region. A total of 453 patients with symptoms of pneumonia and 90 controls with no history of lower respiratory tract infections were included in the study. A duplex polymerase chain reaction (PCR) targeting 543 bp region of P1 adhesin gene of M. pneumoniae and 375 bp region of macrophage infectivity potentiator (mip) gene of L. pneumophila was standardized for simultaneous detection of these atypical pathogens. Respiratory secretions, blood, and urine samples were collected from each patient and control and were subjected to duplex PCR, culture and serology for M. pneumoniae and L. pneumophila. Urine samples were subjected for detecting L. pneumophila antigen. Among the 453 patients investigated for M. pneumoniae, 52 (11.4%) were positive for IgM antibodies, 17 were positive by culture, and seven tested positive by PCR (P1 gene). Similarly for L. pneumophila, 50 cases (11%) were serologically positive for IgM antibodies, one was positive by PCR (mip gene) and urine antigen detection. A total of eight samples were positive by duplex PCR for M. pneumoniae P1 gene (N = 7) and L. pneumophila mip gene (N = 1). Of the 90 controls, two samples (2.2%) showed IgM positivity, and 15 (16.7%) showed IgG positivity for M. pneumoniae. For L. pneumophila, three samples (3.3%) tested positive for IgM, and 12 (13.3%) tested positive for IgG antibodies. The study findings indicate the presence of M. pneumoniae and L. pneumophila in our geographical region, and a combination of laboratory approaches including PCR, culture, and serology is required for effective detection of these agents.
Journal of Clinical Microbiology, 2005
This study describes the development and evaluation of a new commercial test, Chlamylege (Argene Inc.), which allows the simultaneous detection in respiratory samples of Chlamydophila pneumoniae, Mycoplasma pneumoniae, and most Legionella species, as well as PCR inhibitors, by using a multiplex PCR and microplate hybridization. The sensitivities of Chlamylege were 1 ؋ 10 ؊3 IFU, 5 ؋ 10 ؊2 color-changing units, and 1 CFU per reaction tube for C. pneumoniae, M. pneumoniae, and Legionella pneumophila, respectively. A cohort of 154 clinical samples from patients with documented respiratory infections was analyzed by the kit, including 2 samples from patients with C. pneumoniae infection, 9 samples from patients with M. pneumoniae infection, 19 samples from patients with Legionella species infection, and 114 samples that tested negative for the three pathogens. All the positive specimens were correctly detected and identified by the Chlamylege kit, and no false-positive result was observed with the negative samples. The kit was then evaluated in a pediatric prospective study that included 220 endotracheal aspirates, and the results were compared with those obtained by three single in-house PCR assays. Four specimens were found to be positive for C. pneumoniae and six were found to be positive for M. pneumoniae by using both strategies. The Chlamylege kit detected two additional samples positive for M. pneumoniae and one additional sample positive for a Legionella species other than L. pneumophila; these three samples were shown to be true positive by other techniques. These overall results demonstrate that the Chlamylege assay is sensitive, specific, and convenient for the rapid detection and identification of atypical pathogens in clinical samples from patients with respiratory infections.
Diagnostic Microbiology and Infectious Disease, 2003
Atypical pathogens such as Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae are an important cause of community-acquired pneumonia. The available detection methods (culture and serology) either lack sensitivity or give only a retrospective diagnosis. In order to improve their detection and quantification in respiratory samples, a real-time multiplex PCR, performed in two separate reactions, was developed for these three pathogens. The comparison of multiplex real-time and conventional PCR assay on 73 respiratory specimens showed an overall agreement of 98.3%, corresponding to 95.8%, 100% and 100% agreement for C. pneumoniae, L. pneumophila and M. pneumoniae, respectively. Clinical application of this multiplex real-time PCR was done on 40 respiratory samples from 38 patients with respiratory tract infections. Of 19 serology-positive patients, 14 were confirmed by the multiplex real-time PCR to be infected by either one of the three pathogens. All samples from serology-negative patients were negative with the multiplex real-time PCR.
Detection and quantification of Legionella pneumophila in water samples using competitive PCR
Canadian Journal of Microbiology, 2006
Legionella pneumonia can be difficult to diagnose. Existing laboratory tests all have shortcomings, especially the ability to diagnose all Legionella spp. at an early stage. Detection of Legionella DNA in serum can be a valuable tool for the diagnosis of Legionnaires' disease (LD). This report describes two patients with LD diagnosed by PCR using serum samples. In addition, quantification of L. pneumophila DNA using real-time PCR during the course of illness was carried out. The results obtained mirrored both the clinical condition and C-reactive protein values during the course of the illness. Quantification of Legionella DNA in serum using real-time PCR could be a valuable tool to monitor the effects of antimicrobial therapy in patients with LD.
Diagnostic Microbiology and Infectious Disease, 1989
We applied monoclonal antibody typing and restriction endonuclease analysis of plasmid DNA to study 28 clinical and 35 environmental (potable water) isolates of Legionella pneumophila serogroup 1 from three hospitals in Iowa between 1981 and 1986. Monoclonal antibody typing employed a panel of seven antibodies and delineated eight different subtypes. Plasmids were present in 57% of the isolates including 12 of 28 (43%) clinical and 25 of 35 (69%) potable water isolates. The plasmids ranged in size from 28 to 98 kilobase pairs and comprised eight distinct subtypes by restriction endonuclease analysis with Eco RI. Combination of monoclonal antibody and restriction endonuclease subtyping (composite subtyping) revealed 19 different composite subtypes of Legionella pneumophila serogroup 1. The most common composite subtype, 09:04, comprised 29% (18 of 63) of the isolates and was only found in clinical and potable water samples from a single pavilion in hospital A during an outbreak of Legionella pneumophila serogroup 1 pneumonia. Aside from this cluster the diversity of composite subtypes of Legionella pneumophila serogroup 1 observed in clinical and potable water sources over the 5-year period was striking. The combination of monoclonal antibody and restriction endonuclease typing resulted in improved strain delineation and a more useful use of epidemiologic markers for Legionella pneumophila serogroup 1.