Molecular determination,serotyping, antibiotic profile and virulence factors of group B Streptococcus isolated from invasive patients at Arabcare Hospital Laboratory,Palestine

BackgroundStreptococcus agalactiae (group B Streptococcus) is beta-hemolytic, catalase negative, gram-positive cocci, recognized as main bacterial pathogen causing infections in newborns, infants, adults, and elderly people around the world. The aim of this study is to investigate group B Streptococcus samples recovered from invasive patients and determine serotype, virulent genes, and antibiotic-resistant profile of Streptococcus agalactiae in Palestine.MethodsA total of 95 group B Streptococcus strains were isolated from neonates, infants, pregnant and non-pregnant women and males at Arabcare Hospital Laboratory, Palestine, between the period of June 2018 and September 2020. Species identification was carried out through cultivation and conventional biochemical tests. A conventional Polymerase Chain Reaction (cPCR) was used to determine the 5 serotypes and virulent genes of the Streptococcus agalactiae strains. The antibiotic resistance test of group B Streptococcus was evaluated using Kirby-Bauer disk susceptibility. Sequencing and BLAST analysis were used to determine the relationship of the isolates in this study to worldwide isolates.ResultsSerotype III (35%) was the major group B Streptococcus strains serotype causing invasive infections in neonates, infants, pregnant and nonpregnant women, and males, followed by serotypes V (19%), Ia, and II (15%), Ib (6%), respectively. All our isolates encoding for surface protein virulent factors, including a highly virulent gene (HvgA) were mostly found in strains isolated from pregnant women (12%). These group B Streptococcus strains exhibited a high rate of resistance to clindamycin (26%). The overall percentage of levofloxacin resistance was 11%, while vancomycin and ampicillin showed higher resistance, at 14.7 and 16% respectively. In addition, the phylogenetic relationship dendrogram illustrates that Streptococcus agalactiae isolated from an invasive patient (newborn) in Palestine was similar to strains found in China and Japan.ConclusionsThe outcomes of this study demonstrate that resistant group B Streptococcus strains are common in Palestine, therefore, evidence-based infection prevention and antibiotic stewardship efforts are necessary.     

Group B Streptococcus detection: comparison of PCR assay and culture as a screening method for pregnant women

Streptococcus agalactiae or group B Streptococcus (GBS) is one of the most important causal agents of serious neonatal infections. Numerous assays have been evaluated for GBS screening in order to validate a fast and efficient method. The aim of this study was to compare the culture technique (established as the gold standard) with the molecular method of polymerase chain reaction (PCR) with specific primers (atr gene). Two hundred and sixty-three samples were analyzed. Vaginal samples were collected, according to the Centers for Disease Control and Prevention (CDC) recommendations, from women over 35 weeks of pregnancy at Hospital de Clínicas de Porto Alegre (HCPA). Two different extraction methods were tested in all samples collected. PCR technique yielded 71 (26.99%) positive results. Sensitivity and specificity for PCR were 100% and 86.88%, respectively. PCR demonstrated a shorter turnaround time than the culture. The molecular methodology proved to be a useful screening for GBS, allowing effective treatment to be initiated in shorter time to prevent newborn infection.     

Comparison of different sampling techniques and of different culture methods for detection of group B streptococcus carriage in pregnant women

Background  

Streptococcus agalactiae (group B streptococcus; GBS) is a significant cause of perinatal and neonatal infections worldwide. To detect GBS colonization in pregnant women, the CDC recommends isolation of the bacterium from vaginal and anorectal swab samples by growth in a selective enrichment medium, such as Lim broth (Todd-Hewitt broth supplemented with selective antibiotics), followed by subculture on sheep blood agar. However, this procedure may require 48 h to complete. We compared different sampling and culture techniques for the detection of GBS.     

Systematic PCR Detection in Culture-negative Osteoarticular Infections

Background

Identification of microorganisms is crucial for the successful treatment of osteoarticular infections. Molecular methods are more sensitive than culture-dependent methods but may suffer from lack of specificity.

Methods

We studied a large series of 3840 bone and joint culture-negative samples collected from 2308 patients hospitalized in Marseille University Hospitals from November 2007 to October 2009. The samples were systematically cultured for 15 days, and conventional broad-range polymerase chain reaction (PCR) (16S rDNA and 18S rDNA) as well as real-time PCR assays targeting human Bglobin, Staphylococcus aureus, and Kingella kingae were realized on one culture-negative specimen.

Results

Specimens from 741 patients (32.1%) tested positive by culture, including 38 in which bacteria grew only after 6 days of incubation. PCR was positive in 141 (9%) culture-negative specimens. Microorganisms identified by PCR were classified into 2 groups: fastidious bacteria (n = 35), mostly anaerobes in adult patients, and K. kingae in children; and nonfastidious bacteria (n = 106), mostly S. aureus (32.7%). A discrepancy between a positive PCR result for S. aureus and a negative culture were explained by previous antibiotherapy in 31.4% of cases. Our study highlights the usefulness of systematic 16S rDNA gene PCR for the diagnosis of bone and joint infections in culture-negative patients, thus enabling the administration of specific antibiotic treatments.

Conclusions

We recommend the use of conventional broad-range PCR for culture-negative bone and joint specimens, as well as S. aureus-specific PCR for adults and K. kingae-specific PCR for children. 18S rDNA PCR should be reserved only for specific cases.     

兔须癣毛癣菌感染样本常规诊断与定量PCR诊断的比较研究

目的为探讨须癣毛癣菌快速,敏感,特异的诊断方法.方法 采集患兔临床样本63例,分别进行培养法,镜检法和定量PCR法诊断,比较这些方法的敏感性,特异性,阳性预测值和阴性预测值.结果 显示样本培养法,镜检法和定量PCR法的阳性率分别为65%,73%和86%;与常规方法(培养法和镜检法)相比,定量PCR法敏感性和阴性预测值均为100%,高于培养法(77.35%和45.45%)和镜检法(86.79%和64.17%),而特异性(90.90%对100%~100%)和阳性预测值(90.90%对100%~100%)两种方法无明显差异.结论 培养法特异,但敏感性差,耗时长,有假阴性;镜检快速,但不特异,敏感性低;而定量PCR快速,敏感,又特异,可以替代镜检用于临床样品的诊断,但不能替代培养法.     

Evaluation of VIASURE Sexually Transmitted Diseases Real Time PCR Detection kit (CerTest Biotec) for the diagnostic of sexually transmitted infections

IntroductionSexually transmitted infections (STIs) are common in our environment, and trends have been increasing in the last few years. Different methods for STIs diagnosis have been applied by microbiology laboratories over years, but real-time PCR has improved this process. Our objective was to evaluate VIASURE Sexually Transmitted Diseases Real-Time PCR Detection kit (CerTestBiotec, S.L.) comparing with the real-time PCR technique used in our laboratory (Allplex? STI7 Essential Assay, Seegene) which was considered as reference assay.MethodsA total of 948 samples from different sites (vaginal, endocervical, urethral, rectal, pharyngeal swabs and urine samples) were analyzed from July to September 2018.ResultsA discordant result was obtained in 4.5% (43 samples). These discrepancies were mainly observed in threshold cycle (Ct) value next to the limit of detection. The k coefficient obtained shows a very high agreement between both methods with k values from 0.92 to 0.99.ConclusionsVIASURE Sexually Transmitted Diseases Real-Time PCR Detection kit provides a very good correlation with Allplex STI7 and therefore, it's a good tool for the diagnostic of STIs. Positive results with Ct value obtained from 35 and low amplification signal should be applied with caution and should be interpreted based on the patient's clinical data.     

Invasive Group B Streptococcal Disease in Non-pregnant Adults

Abstract Streptococcus agalactiae, commonly referred as group B Streptococcus (GBS), is a major cause of neonatal sepsis and infections in pregnant women. However, the number of invasive infections in non-pregnant adults is growing. Elderly patients and those with chronic underlying conditions, such as diabetes mellitus or compromised immune defence, are at increased risk of invasion. The spectrum of clinical manifestations is broad and includes necrotizing fasciitis and toxic shock syndrome. Although, primary bacteremia and skin and soft-tissue infections are the most frequently reported diagnosis. This article reviews the epidemiology, pathogenesis and treatment of invasive GBS disease in non-pregnant adults, with an emphasis on skin and soft-tissue infections.     

Identification of Streptococcus pneumoniae lytA,plyA and psaA genes in pleural fluid by multiplex real-time PCR

Introduction

The aim was to evaluate the utility of a multiplex real-time PCR to detect Streptococcus pneumoniae lytA, plyA and psaA genes in pleural fluid (PF).

Distribution of serotypes and evaluation of antimicrobial susceptibility among human and bovine Streptococcus agalactiae strains isolated in Brazil between 1980 and 2006

Streptococcus agalactiae is a common agent of clinical and subclinical bovine mastitis and an important cause of human infections, mainly among pregnant women, neonates and nonpregnant adults with underlying diseases. The present study describes the genetic and phenotypic diversity among 392 S. agalactiae human and bovine strains isolated between 1980 and 2006 in Brazil. The most prevalent serotypes were Ia, II, III and V and all the strains were susceptible to penicillin, vancomycin and levofloxacin. Resistance to clindamycin, chloramphenicol, erythromycin, rifampicin and tetracycline was observed. Among the erythromycin resistant strains, mefA/E, ermA and, mainly, ermB gene were detected, and a shift of prevalence from the macrolide resistance phenotype to the macrolide-lincosamide-streptogramin B resistance phenotype over the years was observed. The 23 macrolide-resistant strains showed 19 different pulsed-field gel electrophoresis profiles. Regarding macrolide resistance, a major concern in S. agalactiae epidemiology, the present study describes an increase in erythromycin resistance from the 80s to the 90s followed by a decrease in the 2000–2006 period. Also, the genetic heterogeneity described points out that erythromycin resistance in Brazil is rather due to horizontal gene transmission than to spreading of specific macrolide-resistant clones.     

Real-time PCR: Benefits for Detection of Mild and Asymptomatic Giardia Infections

The majority of Giardia infections are transmitted by the fecal-oral route and cause giardiasis. Children who live in crowded conditions or low socio-economic areas are the risk group for Giardia infection. Interestingly, most of them are asymptomatic or only mildly infected and can shed the Giardia cysts in the environment. Thus, the diagnosis of Giardia infection in asymptomatic or mild infection plays an important role in achieving control of Giardia duodenalis transmission. The objective of this study was to examine parasitic infections using microscopy and to develop a real-time PCR method for detection of Giardia infection in the stool samples of children living on the Thai-Myanmar border. Both species-specific primers and fluorescent labeled G. duodenalis probe were designed using small-subunit ribosomal RNA (ssrRNA). The results showed that 10 (7.69%) and 40 (30.77%) of 130 stool samples were positive for G. duodenalis by microscopy and real-time PCR respectively. Only 3 out of 9 liquid stools revealed G. duodenalis positive using microscopy, but all of them were G. duodenalis-positive using real-time PCR. The detection limit of real-time PCR for G. duodenalis was 0.1 pg/25 µl reaction. It can detect both mild and asymptomatic Giardia infections in children living on the Thai-Myanmar border.     

Schistosoma mansoni in schoolchildren in a Madagascan highland school assessed by PCR and sedimentation microscopy and Bayesian estimation of sensitivities and specificities

Madagascar is an endemic area for schistosomiasis, but recent prevalence data are scarce. We investigated stool samples of 410 children aged 4–18 years from a combined primary and secondary school in a Madagascan highland village near Ambositra in order to assess the prevalence of Schistosoma mansoni using microscopy and real-time polymerase chain reaction (PCR). A high prevalence of S. mansoni of 77.1% was detected by PCR, while only 15.2% of microscopic examinations of sedimentation-enriched stools were positive. We estimated the sensitivity and specificity of stool sedimentation microscopy (19.7% and 98.8%) and of PCR (98.9% and 89.3%) using a Bayesian approach for two dependant tests in one population without a reference standard. Our Bayesian posterior estimate of the prevalence is 80.2%. Simple sedimentation technique misses about 4/5 of all PCR-confirmed infections and is insufficient to determine the prevalence of S. mansoni. A survey comparing PCR with a classical standard technique (KatoKatz) is desirable.     

Epidural abscess caused by <Emphasis Type="Italic">Streptococcus milleri</Emphasis> in a pregnant woman

Background  

Bacteria in the Streptococcus milleri group (S. anginosus, S. constellatus, and S. intermedius) are associated with bacteremia and abscess formation. While most reports of Streptococcus milleri group (SMG) infection occur in patients with underlying medical conditions, SMG infections during pregnancy have been documented. However, SMG infections in pregnant women are associated with either neonatal or maternal puerperal sepsis. Albeit rare, S. milleri spinal-epidural abscess in pregnancy has been reported, always as a complication of spinal-epidural anesthesia. We report a case of spinal-epidural abscess caused by SMG in a young, pregnant woman without an antecedent history of spinal epidural anesthesia and without any underlying risk factors for invasive streptococcal disease.     

Evaluation of real-time PCR for Strongyloides stercoralis and hookworm as diagnostic tool in asymptomatic schoolchildren in Cambodia

Diagnosis of soil-transmitted helminths such as Strongyloides stercoralis and hookworms (Ancylostoma duodenale and Necator americanus) is challenging due to irregular larval and egg output in infected individuals and insensitive conventional diagnostic procedures. Sensitive novel real-time PCR assays have been developed. Our study aimed to evaluate the real-time PCR assays as a diagnostic tool for detection of Strongyloides spp. and hookworms in a random stool sample of 218 asymptomatic schoolchildren in Cambodia.Overall prevalence of 17.4% (38/218) and 34.9% (76/218) were determined by real-time PCR for S. stercoralis and hookworms, respectively. Sensitivity and specificity of S. stercoralis specific real-time PCR as compared to the combination of Baermann/Koga Agar as gold standard were 88.9% and 92.7%, respectively. For hookworm specific real-time PCR a sensitivity of 78.9% and specificity of 78.9% were calculated. Co-infections were detectable by PCR in 12.8% (28/218) of individuals.S. stercoralis real-time PCR applied in asymptomatic cases showed a lower sensitivity compared to studies undertaken with symptomatic patients with the same molecular tool, yet it proved to be a valid supplement in the diagnosis of STH infection in Cambodia.     

Cervical ureaplasma urealyticum colonization: comparison of PCR and culture for its detection and association with preterm birth

We developed a 16S ribosomal (r) RNA gene-based PCR assay specific for Ureaplasma urealyticum and compared it with culture. We also wanted to assess the role of cervical U. urealyticum colonization in preterm births. Cervical swabs from 100 women with preterm contractions and from 50 asymptomatic pregnant women were collected and analyzed using PCR. The PCR and culture methods were compared using the samples from the asymptomatic patients. PCR and culture were equally effective at detecting U. urealyticum. Cervical colonization correlated with preterm delivery, with rates of 71% and 37% for those delivering preterm and those with term delivery, respectively (p = 0.008). The relative risk of preterm delivery if colonized with U. urealyticum was 3.34. 16S rRNA gene-based PCR proved to be a useful tool for detecting U. urealyticum compared to culture. Lower genital tract colonization with U. urealyticum was associated with preterm birth.     

Real-time PCR is more specific than conventional PCR for induced sputum diagnosis of Pneumocystis pneumonia in immunocompromised patients without HIV infection

Background and objective:   The diagnosis of Pneumocystis pneumonia (PCP) is based on microscopic examination of respiratory specimens. PCP patients without AIDS have a lower burden of P. jiroveci than those with AIDS, which leads to difficulty in detecting the organisms. Although conventional PCR (c-PCR) has been used to detect the DNA, it is frequently positive in patients with colonization. Real-time PCR (r-PCR), a method to detect the DNA quantitatively, might be helpful in distinguishing between infection and colonization. We investigated the utility of real-time PCR in the diagnosis of PCP in non-AIDS patients.
Methods:   Induced sputum samples obtained from 86 non-HIV immunocompromized patients with clinical symptoms of pulmonary infection were evaluated for the presence of Pneumocystis jiroveci -specific DNA using c-PCR and r-PCR. The diagnosis of PCP was confirmed by typical clinical and radiological findings and response to treatment.
Results:   Of the 86 patients, 17 were diagnosed as having PCP. Twenty-eight samples were positive for c-PCR, but the false-positive rate was high (46.4%). Sensitivity, specificity and positive predictive values (PPV) of c-PCR were 88.2%, 81.2% and 53.6%, respectively. Concentrations of the DNA detected by r-PCR were significantly higher in PCP patients than in non-PCP patients. Using 30 copies per tube as a cut-off value for the diagnosis of PCP, the sensitivity (82.4%) of r-PCR was almost equal to c-PCR. Notably, its specificity and PPV were higher than c-PCR (98.6% and 93.3%, respectively).
Conclusions:   r-PCR on induced sputum is more useful for diagnosing PCP than c-PCR in non-HIV immunocompromized patients, especially in terms of distinguishing between colonization and infection.     

Development and validation of a Pneumocystis jirovecii real-time polymerase chain reaction assay for diagnosis of Pneumocystis pneumonia

BACKGROUND:Pneumocystis jirovecii (PJ), a pathogenic fungus, causes severe interstitial Pneumocystis pneumonia (PCP) among immunocompromised patients. A laboratory-developed real-time polyermase chain reaction (PCR) assay was validated for PJ detection to improve diagnosis of PCP.METHODS:Forty stored bronchoalveolar lavage (BAL) samples (20 known PJ positive [PJ+] and 20 known PJ negative [PJ−]) were initially tested using the molecular assay. Ninety-two sequentially collected BAL samples were then analyzed using an immunofluorescence assay (IFA) and secondarily tested using the PJ real-time PCR assay. Discrepant results were resolved by retesting BAL samples using another real-time PCR assay with a different target. PJ real-time PCR assay performance was compared with the existing gold standard (ie, IFA) and a modified gold standard, in which a true positive was defined as a sample that tested positive in two of three methods in a patient suspected to have PCP.RESULTS:Ninety of 132 (68%) BAL fluid samples were collected from immunocompromised patients. Thirteen of 92 (14%) BALs collected were PJ+ when tested using IFA. A total of 40 BAL samples were PJ+ in the present study including: all IFA positive samples (n=13); all referred PJ+ BAL samples (n=20); and seven additional BAL samples that were IFA negative, but positive using the modified gold standard. Compared with IFA, the PJ real-time PCR had sensitivity, specificity, and positive and negative predictive values of 100%, 91%, 65% and 100%, respectively. Compared with the modified gold standard, PJ real-time PCR had a sensitivity, specificity, and positive and negative predictive values of 100%.CONCLUSION:PJ real-time PCR improved detection of PJ in immunocompromised patients.     

Shaping a bacterial genome by large chromosomal replacements, the evolutionary history of Streptococcus agalactiae

Bacterial populations are subject to complex processes of diversification that involve mutation and horizontal DNA transfer mediated by transformation, transduction, or conjugation. Tracing the evolutionary events leading to genetic changes allows us to infer the history of a microbe. Here, we combine experimental and in silico approaches to explore the forces that drive the genome dynamics of Streptococcus agalactiae, the leading cause of neonatal infections. We demonstrate that large DNA segments of up to 334 kb of the chromosome of S. agalactiae can be transferred through conjugation from multiple initiation sites. Consistently, a genome-wide map analysis of nucleotide polymorphisms among eight human isolates demonstrated that each chromosome is a mosaic of large chromosomal fragments from different ancestors suggesting that large DNA exchanges have contributed to the genome dynamics in the natural population. The analysis of the resulting genetic flux led us to propose a model for the evolutionary history of this species in which clonal complexes of clinical importance derived from a single clone that evolved by exchanging large chromosomal regions with more distantly related strains. The emergence of this clone could be linked to selective sweeps associated with the reduction of genetic diversity in three regions within a large panel of human isolates. Up to now sex in bacteria has been assumed to involve mainly small regions; our results define S. agalactiae as an alternative paradigm in the study of bacterial evolution.     

Application of real-time PCR for the detection of Strongyloides spp. in clinical samples in a reference center in Spain

Strongyloidiasis is one of the major intestinal helminthic infections in humans with a worldwide distribution, affecting especially tropical and subtropical regions. This disease can occur without any symptoms or as a potentially fatal hyperinfection or disseminated infection. Definitive diagnosis of Strongyloides stercoralis infection relies mainly on demonstration of larvae in stool, but at present there is no gold standard for this diagnosis. Our main objective was to evaluate a real-time PCR targeting the 18S rRNA gene of Strongyloides spp. and to compare it with routine parasitological methods. DNA from Strongyloides venezuelensis was used to optimize PCR protocols obtaining an analytical sensitivity of 0.1 pg of parasite DNA per sample. Sensitivity and specificity of real-time PCR on fecal samples from 231 patients screened for suspected strongyloidiasis attending two hospitals in Madrid were 93.8% and 86.5%, respectively. No significant differences were found when comparing Ct-values of positive PCR between parasitological positive and negative samples. This study showed that real-time PCR is an effective tool for diagnosing strongyloidiasis and could be applied in association with parasitological methods in epidemiological studies in endemic areas. It would be also important to assess its performance in immunocompromised populations who are at risk of fatal disease.     

A comparison between the efficiency of the Xpert MTB/RIF assay and nested PCR in identifying Mycobacterium tuberculosis during routine clinical practice

Objectives

Polymerase chain reaction (PCR) for the detection of Mycobacterium tuberculosis (MTB) is more sensitive, specific, and rapid than the conventional methods of acid-fast bacilli (AFB) smear and culture. The aim of this study was to determine if the Xpert MTB/rifampicin (RIF) assay had additional advantages over nested PCR for the detection of MTB in a geographical area with intermediate tuberculosis (TB) incidence.

Methods

Between February and December 2013, the Xpert MTB/RIF assay and MTB nested PCR, as well as AFB smear and culture, were simultaneously performed on 198 clinical samples (160 pulmonary and 38 non-pulmonary specimens) collected from 171 patients hospitalized at Hallym University Medical Center for possible TB. The accuracy of the diagnosis of MTB culture-positive TB and the turnaround time of reporting laboratory results were calculated and compared. Rifampin resistance by the Xpert MTB/RIF assay was reviewed with that of conventional drug susceptibility testing (DST).

Results

The sensitivity, specificity, and positive and negative predictive values of the Xpert MTB/RIF assay and MTB nested PCR for diagnosis of MTB culture-positive pulmonary TB were 86.1% vs. 69.4% (P=0.1563), 97.8% vs. 94.1% (P=0.2173), 91.2% vs. 75.8% (P=0.1695), and 96.4% vs. 92.0% (P=0.2032), respectively. The median turnaround times of the Xpert MTB/RIF assay and MTB nested PCR were 0 [0-4] days and 4 [1-11] days, respectively (P<0.001). Two cases of rifampin resistance, as determined by the Xpert MTB/RIF assay, were found to be multi-drug resistant (MDR) pulmonary TB by DST.

Conclusions

The Xpert MTB/RIF assay seemed to be sensitive, specific, and comparable to nested PCR for identifying MTB among clinically suspected TB patients, and the assay can be valuable in giving a timely identification of resistance to rifampin.     

Influence of 16S ribosomal RNA gene polymerase chain reaction and sequencing on antibiotic management of bone and joint infections

INTRODUCTION:

Amplification of the 16S ribosomal RNA gene by polymerase chain reaction (PCR) followed by analysis of generated sequences can be an important adjunct to conventional cultures.

OBJECTIVE:

To determine how the results of this approach influence physicians’ decisions regarding the management of bone and joint infections.

METHOD:

Clinical and laboratory findings of patients seen at the Queen Elizabeth II Health Sciences Centre (Halifax, Nova Scotia) between December 2005 and September 2009 were reviewed. Patients who had negative cultures but likely or possible bone and joint infections were further evaluated using 16S rRNA PCR. The impact of the 16S rRNA PCR result on antibiotic management was evaluated and it was assessed whether untreated patients with negative 16S rRNA PCR subsequently presented with infections, suggesting a false-negative result.

RESULT:

A total of 36 patients (mean age 62 years) were reviewed. Thirty-two patients were evaluated by infectious disease consultants; of these, 20 were considered likely to have infections. Seventeen patients were admitted with suspected prosthetic joint infections. Twenty-nine patients received antimicrobial treatment before the sample for the 16S rRNA PCR assay was obtained. Of the 36 patients, 26 (72.2%) were treated appropriately with modifications to their antibiotic regimen in response to the 16S rRNA PCR assay results. Antimicrobials were discontinued for 19 patients based on negative PCR assay and, in seven patients, antibiotics were changed based on a positive result. There were no relapses among patients with negative PCR assay in whom antibiotics were discontinued.

CONCLUSION:

16S ribosomal RNA gene PCR and sequencing is a valuable tool in the guidance of antimicrobial therapy for bone and joint infections.