Enhancing sensitivity of qPCR assays targeting Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae by using a mutant Taq DNA polymerase.

AIMS: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae are important causes of bacterial meningitis. In this study, the DNA binding site of the wild type Taq DNA polymerase was modified to produce a mutant enzyme with enhanced DNA affinity and PCR performance. The engineered and the wild type enzymes were integrated into qPCR-based assays for molecular detection of S. pneumoniae, N. meningitidis, H. influenzae, and serogroups and serotypes of these three pathogens. METHODS: Bio-Speedy® Bacterial DNA Isolation Kit (Bioeksen R&D Technologies, Turkiye) and 2× qPCR-Mix for hydrolysis probes (Bioeksen R&D Technologies, Turkiye) and CFX96 Instrument (Biorad Inc., USA) were used for all molecular analyses. Spiked negative clinical specimens were tested using the developed qPCR assays and the culture-based conventional methods for the analytical performance evaluation. RESULTS: All qPCR assays did not produce any positive results for the samples spiked with potential cross-reacting bacteria. Limit of detection (LOD) of the assays containing the mutant enzyme was 1 genome/reaction (10 cfu/mL sample) which is at least 3 times lower than the previously reported LOD levels for DNA amplification based molecular assays. LODs for the spiked serum and cerebrospinal fluid (CSF) samples decreased 2.3-4.7 and 1.2-3.5 times respectively when the mutant enzyme was used instead of the wild type Taq DNA polymerase. CONCLUSIONS: It is possible to enhance analytical sensitivity of qPCR assays targeting the bacterial agents of meningitis by using an engineered Taq DNA polymerase. These qPCR-based assays can be used for direct detection and serogrouping / serotyping of S. pneumoniae, N. meningitidis and H. influenzae at concentrations close to the lower limit of medical decision point.

Development of a new multiplex real-time PCR assay for rapid screening of hospital-acquired infection agents.

AIMS: A new multiplex real-time PCR (qPCR) assay was developed to detect antibiotic-resistant hospital-acquired infectious agents in nasal and rectal swab samples in 1.5 h without the need for nucleic acid extraction. METHODS: Spiked negative clinical specimens were used for the analytical performance evaluation. Double-blind samples were collected from 1788 patients to assess the relative clinical performance of the qPCR assay to the conventional culture-based methods. Bio-Speedy® Fast Lysis Buffer (FLB) and 2× qPCR-Mix for hydrolysis probes (Bioeksen R&D Technologies, Istanbul, Turkey) and LightCycler® 96 Instrument (Roche Inc., Branchburg, NJ, USA) were used for all molecular analyses. The samples were transferred into 400 L FLB, homogenized and immediately used in qPCRs. The target DNA regions are vanA and vanB genes for vancomycin-resistant Enterococcus (VRE); blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-23, blaOXA-48, blaOXA-58 genes for carbapenem-resistant Enterobacteriaceae (CRE); and mecA, mecC and spa for methicillin-resistant Staphylococcus aureus (MRSA). RESULTS: No qPCR tests produced positive results for the samples spiked with the potential cross-reacting organisms. The limit of detection (LOD) of the assay for all targets was 100 colony-forming unit (cfu)/swab-sample. Results of the repeatability studies in two different centers were in 96%-100% (69/72-72/72) agreement. The relative specificity and sensitivity of the qPCR assay were respectively 96.8% and 98.8% for VRE; 94.9% and 95.1% for CRE; 99.9% and 97.1% for MRSA. CONCLUSIONS: The developed qPCR assay can screen antibiotic-resistant hospital-acquired infectious agents in infected/colonized patients with an equal clinical performance to the culture-based methods.

[A Case of Oropharyngeal Tularemia Mimicking Lymphoma During Pregnancy]. / Gebelikte Lenfomayi Taklit Eden Bir Orofarengeal Tularemi Olgusu.

Tularemia is a zoonotic bacterial infectious disease caused by a gram-negative coccobacillus namely Francisella tularensis. In humans, disease leads to several different clinical forms (ulceroglandular, glandular, oculoglandular, respiratory, typhoidal and oropharyngeal). Since the main mode of transmission of the disease to humans in Türkiye is by drinking water contaminated with F.tularensis, the oropharyngeal form is the most common clinical manifestation. Since tularemia cases with pregnancy are rare, the literatüre about maternal and fetal complications of tularemia is sparse. In this report, a case of oropharyngeal tularemia mimicking lymphoma during pregnancy was presented. A 33-year-old 11-week pregnant patient living in a village in Sivas province admitted to the infectious diseases and clinical microbiology outpatient clinic with the complaint of swelling in the neck region that continued for six days. The patient, who was engaged in animal husbandry stated that she consumed raw milk and admitted to the otorhinolaryngology outpatient clinic of a hospital 10 days ago with the complaints of fever, chills, and sore throat. She stated that her complaints did not regress with the amoxicillin-clavulanate treatment recommended by her doctor and she noticed the swelling in her neck on the 4th day of the treatment. Upon further questioning, it was understood that the patient had a history of consumption of unchlorinated spring water. Her vital signs were normal and physical examination revealed non-fluctuant lymph nodes with the largest of 5 x 2 cm in the right posterior cervical region, and 3 x 2 cm in the left. Laboratory tests revealed a blood leukocyte count of 13.32 x 103/mm3 (75% granulocytes), a blood hemoglobin of 11.4 g/dL, an erythrocyte sedimentation rate of 45 mm/hour, and C-reactive protein of 90 mg/L. A non-contrast MRI examination revealed wall thickening of the nasopharynx and enlarged lymph nodes which were suspicious for lymphoma with significant diffusion restriction on diffusionweighted images. As the past medical history and clinical findings were suggestive for tularemia, the microagglutination test (MAT) was studied, but it was reported as negative with a titer at 1/80. Since the patient's complaints continued and tularemia cases were encountered in our region in the past years, the repeated MAT after two weeks was reported as positive with a titer at 1/320. An oropharyngeal form of tularemia was diagnosed and oral ciprofloxacin (2 x 750 mg) was given for three weeks by starting at the 14th gestational week. Lymphoma was excluded by histopathological examination of the fine needle aspiration biopsy performed on the patient's cervical lymph nodes, but the biopsy sample was compatible with granulomatous diseases. Histopathological findings of diagnostic biopsies of the larynx and nasopharynx were reactive. A healthy male baby, 2425 grams, 47 cm, was delivered by cesarean section from the patient who presented with labor contractions at the 37th week of pregnancy. There was no sign of congenital infection in the newborn. The patient and the baby were followed up to the end of one year and no abnormality was found. The evaluation of 17 cases reported in the literatüre including this case, suggest that tularemia may progress to involve serious obstetric complications during pregnancy, such as abortion, premature birth and intrauterine fetal death when appropriate and effective antibiotic treatment is not given.

Brucella melitensis and Brucella abortus genotyping via real-time PCR targeting 21 variable genome loci.

Brucella melitensis and Brucella abortus account for almost all cases of brucellosis in Turkish population. We developed a fourplex quantitative real-time PCR (qPCR) assay for the electrophoresis-free, rapid and cost-effective differentiation of B. abortus and B. melitensis from the other Brucella spp. The 4-plex species differentiation assay was combined with a qPCR assay targeting 17 different single nucleotide polymorphism (SNP) loci in Brucella genomes. This combination resulted in a 21 Variable Genome Loci (21-VGL) qPCR assay for high resolution genotyping of B. abortus and B. melitensis. A total of 486 Brucella was analyzed using the qPCR assay to create a 21-VGL profile database. The database contained the profiles of 55 B. abortus, 352 B. melitensis, 3 B. ceti, 6 B. neotomae, 7 B. ovis, 6 B. pinnipedialis, 44 B. suis and 13 B. canis strains. The 21-VGL Brucella genotyping clearly distinguished B. abortus, B. melitensis, B. neotomae and B. ovis. The 21-VGL approach could not distinguish B. pinnipedialis from B. ceti and some B. suis genotypes from B. canis. The results revealed that more than 99% of the Brucella isolates in Turkey were B. melitensis and 21-VGL genotyping can be reduced to 8-VGL B. melitensis genotyping without any loss of genotyping resolution. To our knowledge, we introduced the fastest and the lowest-cost B. abortus and B. melitensis genotyping and species differentiation methodology in the literature.

[Optimization of real-time multiplex polymerase chain reaction for the diagnosis of acute bacterial meningitis and Neisseria meningitidis serogrouping]. / Akut bakteriyel menenjit tanisi ve Neisseria meningitidis serogruplandirmasinda gerçek zamanli multipleks polimeraz zincir reaksiyonunun optimizasyonu.

Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae type B are responsible for about 80-85% of acute bacterial meningitis cases among adults and in our country it is mandatory to report bacterial meningitis cases caused by these agents. In recent years, disease control programs have focused especially on these three pathogens in our country. It is very important to know the causative agent of meningitis for prevention/control measures, choice of treatment regimen, and prediction of the severity and the course of the disease. Rapid diagnosis of meningococcal meningitis is especially vital. In addition, the identification of N.meningitidis serogroups in the community is also important for the decision of the type and combination of vaccine to be administered. The aim of this study was to optimize the real-time multiplex polymerase chain reaction (Rt-multiplex PCR) for the diagnosis of the disease and serogrouping of N.meningitidis in clinical samples in a direct, quick and reliable way. In this study, three Rt-multiplex PCR assays were optimized and standardized for the detection of N.meningitidis, H.influenzae and S.pneumoniae (NHS mix) and for the serogrouping of N.meningitidis (BCY mix and AWX mix) in our laboratory. Sensitivity of the Rt-multiplex PCR method was detected by reference strains and simulation studies. Forty three samples (41 cerebrospinal fluid (CSF), 1 serum and 1 clinical isolate) with the suspicion of acute bacterial meningitis and six nasopharyngeal isolates sent to National Reference Laboratory for Molecular Microbiology between July 2012-May 2014 were included in the present study. All samples were examined with the Rt-multiplex PCR methods. Clinical isolates were studied by both conventional and Rt-multiplex PCR methods. Oxidase, catalase test, Gram stain, API-NH and agglutination tests with specific antisera were performed in the National Respiratory Pathogens Reference Laboratory. The detection limit of the method, which was optimized and standardized in our laboratory was determined as 102 cfu/ml. The CT (threshold cycle) value in this dilution was detected approximately as 35. N.meningitidis was detected in 14 of the 41 CSF samples by the NHS mix. However, only 10 of the positive samples could be typed with BCY mix and AWX mix. Eight (80%) of them were serogroup B, one of each was (10%) serogroup A and serogroup W135, respectively. All the isolates (six nasopharyngeal and one clinical specimen) were identified as N.meningitidis serogroup B by Rt-multiplex PCR and the isolates were also confirmed by conventional methods. The CSF specimens with CT value > 35 could not be typed. We concluded that the Rt-multiplex PCR method is a rapid and reliable test for the direct diagnosis of acute bacterial meningitis due to NHS and serogrouping of N.meningitidis. Rapid diagnosis plays an important role for the treatment and control of the disease, and serogrouping of the agent plays an important role in terms of prevention/control and vaccination policies.

[Screening of Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis in throat swab specimens of children with upper respiratory tract infections]. / Üst solunum yolu enfeksiyonu bulgulari ile basvuran çocuklarin bogaz sürüntü örneklerinde Corynebacterium diphtheriae, Corynebacterium ulcerans ve Corynebacterium pseudotuberculosis suslarinin arastirilmasi.

Although a significant decrease has been reported in the incidence of diphteria in many regions of the world following the routine diphtheria immunization programs, the emergence of new cases indicated that toxigenic strains are still circulating in the community. Diphtheria vaccine does not provide protection against asymptomatic carriage and colonization of non-toxigenic Corynebacterium diphtheriae. It is a known fact that invasive infections may arise from non-toxigenic C.diphtheriae strains that the non-toxigenic strains can become toxigenic strains leading to diphteria. It is also known that there is a risk of diphteria outbreaks due to decreased antitoxin level and inadequate adult immunization programs. In our country, there is no routine surveillance of toxigenic and non-toxigenic C.diphtheriae. In the present study we aimed to investigate the presence of C.diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis in children presenting with the symptoms of upper respiratory tract infections that might be confused with those moderate diphteria, in order to highlight the requirement of microbiological surveillance and to create awareness about these microorganisms among public health experts, microbiologists and clinicians. Throat swab specimens were obtained from children who were admitted to the pediatric outpatient clinics, in Dr. Sami Ulus Obstectrics, Children Health and Diseases Educational and Research Hospital, with upper respiratory tract infections between 1 February 2016-22 March 2016. The specimens were inoculated in 5% sheep blood agar plates. The plates that were incubated in appropriate conditions, were evaluated for Group A beta hemolytic streptococcocci. Subsequently, culture plates were sent to the Public Health Institution of Turkey, National Respiratory Pathologens Reference Laboratories for the investigation of the presence of C.diphtheriae, C.ulcerans and C.pseudotuberculosis. The growth in each plate were collected with a sterile swab and inoculated in tryptic soy broth. Following 2 hours of incubation at 37oC, subcultures were inoculated in cystine-tellurite-blood agar (CTBA) and 5% sheep-blood agar plates; after an overnight incubation tellurite-reducing colonies were inoculated in Tinsdale agar plates. The suspected colonies with positive cystinase activity were identified by conventional methods and also with Coryne API (Biomerieux, France) systems. Toxicity tests (ELEK, PCR) were performed to investigate whether the C.diphtheriae strains were producing toxins. A total of 500 patients were involved in the study. Of these 260 (52%) were girls and 240 (48%) were boys with a mean age of 76 (range, 21-213) months. All patients except one were fully vaccinated with boosters. Most common presenting symptoms of the patients were fever (19.8%), sore throat (52.6%), cough (49.2%), tonsillar hyperemia (97.6%), presence of crypt (24.6%), and membrane over tonsils (1%). Group A beta-hemolytic streptococcocci were detected in the throat swab cultures of 66 (%13.2) patients. Genotypically toxin negative C.diphtheriae biovar gravis was identified in the throat swab cultures of 3 patients (2 girls and 1 boy). The tonsils were hyperemic and hypertrophic in all the patients with C.diphtheriae biovar gravis. C.ulcerans and C.pseudotuberculosis were detected in none of the patients. It is considered that similar regular cross-sectional studies or routine screening programs are expected to raise awareness about this forgotten microorganism both epidemiologically and microbiologically.

Panton-Valentine leucocidin gene carriage among Staphylococcus aureus strains recovered from skin and soft tissue infections in Turkey.

OBJECTIVES: Regardless of methicillin resistance, Panton-Valentine leucocidin (PVL)-positive Staphylococcus aureus isolates are associated with various types of infections and outbreaks. Limited data exist about the PVL content of S. aureus strains in Turkey. In this multicentre study, we aimed to assess the PVL positivity and antimicrobial susceptibilities of S. aureus isolates recovered from skin and soft tissue samples of both community and nosocomial origin in the study period, 2007-08. METHODS: Two hundred and forty-two [92 community-acquired (CA) and 150 hospital-acquired (HA)] isolates were included in the study. Analysis of mecA and PVL was carried out using PCR. All isolates underwent susceptibility testing according to the CLSI. RESULTS: Out of 242 isolates, 77 were mecA positive. PVL was not found among methicillin-resistant S. aureus (MRSA) isolates, but 8 (5.3%) HA methicillin-susceptible S. aureus (MSSA) and 14 (15.2%) CA-MSSA, mostly isolated from furuncles (71.4%), were positive for PVL. Among PVL-positive strains, the penicillin resistance rate was 90.9%. Low resistance rates, <10%, were detected for erythromycin, fusidic acid and co-trimoxazole. PVL-positive strains showed higher rates of susceptibility to erythromycin, gentamicin and rifampicin than negative isolates. CONCLUSIONS: Based on the findings of this study, infection related to PVL-carrying CA-MRSA is not at an alarmingly high level, but population-based surveillance studies should be done to determine the real status.