Contribution of a broad range polymerase chain reaction to the diagnosis of osteoarticular infections caused by Kingella kingae: description of twenty-four recent pediatric diagnoses

BACKGROUND: Microbiologic diagnosis of septic arthritis and osteomyelitis in children is hindered by the less than optimal yield of blood and osteoarticular fluid cultures. PATIENTS AND METHODS: All patients admitted to a pediatric unit for osteoarticular infections (OAI) between January 2001 and February 2004 were enrolled in this prospective study. Osteoarticular fluid and biopsy samples that were negative by conventional culture were tested by polymerase chain reaction (PCR) with universal 16S ribosomal DNA primers. RESULTS: We enrolled 171 children. Culture was positive in 64 cases (37.4%), yielding Kingella kingae in 9 cases. The 107 culture-negative specimens were tested by 16S ribosomal DNA PCR. Fifteen samples (14%) were positive, all for Kingella DNA sequences. K. kingae was the second cause of OAI in this population (30.4%), after Staphylococcus aureus (38%). Patients with Kingella infection diagnosed by culture (9 cases) did not differ from those diagnosed by PCR (15 cases) in terms of their clinical characteristics (including prior antibiotic therapy). The characteristics of the 24 children with arthritis (n = 17) or osteomyelitis (n = 7) were similar to those reported elsewhere. Fever (>38 degrees C) and symptom onset shortly before hospitalization (median, 4.5 days) were significantly associated with arthritis. CONCLUSION: Use of molecular diagnostic methods increases the identification of K. kingae in osteoarticular infections.     

Kingella kingae: an emerging pathogen in young children

Kingella kingae is being recognized increasingly as a common etiology of pediatric osteoarticular infections, bacteremia, and endocarditis, which reflects improved culture methods and use of nucleic acid-amplification techniques in clinical microbiology laboratories. K kingae colonizes the posterior pharynx of young children and is transmitted from child to child through close personal contact. Day care attendance increases the risk for colonization and transmission, and clusters of K kingae infections among day care center attendees have been reported. Key virulence factors in K kingae include type IV pili and a potent RTX toxin. In previously healthy children, >95% of K kingae infections are diagnosed between the ages of 6 and 48 months. Among children with underlying medical conditions, K kingae disease may occur at older ages as well. The clinical presentation of K kingae disease is often subtle and may be associated with normal levels of acute-phase reactants, which underscores the importance of a high index of suspicion. K kingae is usually susceptible to ?-lactam antibiotics, and infections typically respond well to medical treatment, with the exception of cases of endocarditis.     

Small risk of osteoarticular infections in children with asymptomatic oropharyngeal carriage of kingella kingae

The aim of this study was to evaluate the absolute risk for children younger than 4 years of age with asymptomatic oropharyngeal carriage of Kingella kingae to sustain an osteoarticular infection. The rate of K. kingae carriage in the oropharyngeal mucosa was 9% among healthy children, and the risk for an asymptomatic carrier to develop an osteoarticular infection due to K. Kingae was estimated to be lower than 1%.     

Comparison of clinical and biologic features of Kingella kingae and Staphylococcus aureus arthritis at initial evaluation

We conducted a retrospective study comparing the presenting clinical and biologic features of 64 children who had septic arthritis caused by Kingella kingae with 26 children who had septic arthritis caused by Staphylococcus aureus. Children with K. kingae septic arthritis were significantly younger than those with S. aureus septic arthritis. Otherwise, there were no significant differences between the 2 groups with respect to fever, location, white blood cell count, synovial fluid cell count, C-reactive protein, or serum fibrinogen. However, the clinical course was significantly better for children with septic arthritis caused by K. kingae as evidenced by shorter hospitalization and fewer adverse events. Presumptive antibiotic therapy for septic arthritis in young infants should take into account both of these pathogens, even in case of mild presentation.     

Imipenem and cilastatin in acute osteomyelitis and suppurative arthritis. Therapy in infants and children

Twenty-five infants and children with acute osteomyelitis (n = 7), suppurative arthritis (n = 11), or both (n = 7) were treated with imipenem and cilastatin sodium. Patients ranged in age from 5 months to 11.3 years. Needle aspiration of infected sites was performed in all patients, and 11 (44%) required further surgical drainage. Imipenem and cilastatin sodium in a dosage of 100 mg/kg/d was used for children 3 years of age or younger, while older ones received 60 mg/kg/d intravenously, divided in four equal doses. Bacterial pathogens were identified in 15 patients (60%): Staphylococcus aureus in five, Haemophilus influenzae b in four, Pseudomonas aeruginosa in two, Streptococcus pneumoniae in one, group A Streptococcus in one, Kingella kingae in one, and Citrobacter amalonaticus in one. All isolates were susceptible to imipenem in vitro. Imipenem and cilastatin therapy was continued for a median of six days followed by treatment with appropriate orally administered antibiotics. Median peak serum bactericidal titers after imipenem and cilastatin infusions were 1:512 for S aureus, 1:32 for H influenzae b, 1:512 for streptococci, and 1:16 for gram-negative rods. All but one patient with P aeruginosa osteomyelitis responded favorably to imipenem and cilastatin. The median duration until resolution of symptoms was six days. Imipenem and cilastatin infusions were well tolerated, and side effects included maculopapular rash in one patient, watery diarrhea in one, and mild transient elevation of alanine aminotransferase levels in three. Because of imipenem and cilastatin's unusually broad spectrum of activity and its relative safety, this drug combination can be used for the initial, empiric therapy of acute bone and joint infections in pediatric patients.     

Empyema: the use of broad range 16S rDNA PCR for pathogen detection.

BACKGROUND: An increase in the incidence of thoracic empyema in children has been reported. The causative pathogen is often unknown as pleural fluid is frequently sterile at the time of culture. The role of unusual organisms is unclear. AIMS: (1) To compare the detection of organisms in pleural fluid from children with empyema using a molecular technique (16S rDNA polymerase chain reaction (PCR)) and bacterial culture. (2) To compare the concordance of organisms identified using the two techniques and the influence of prior antibiotic treatment on positive detection rate. METHODS: Pleural fluid from children admitted with empyema between January 2000 and February 2002 was cultured and additionally analysed using broad range 16S rDNA PCR. RESULTS: Pleural fluid was cultured from 32 patients, aged 1 month-16 years. Median duration of previous antibiotic therapy was 8 days (range 1-42 days). Six samples were culture positive and 22 were PCR positive. A causal organism was detected by PCR alone, after considering results from the local hospital, in 14 patients. There was complete concordance in organisms cultured and detected by PCR. Additional organisms detected by PCR were predominantly S pneumoniae, S pyogenes, and anaerobes. CONCLUSIONS: Analysis of pleural fluid by broad range 16S rDNA PCR in addition to culture, increases organism identification in empyema.     

Empyema: the use of broad range 16S rDNA PCR for pathogen detection

Background: An increase in the incidence of thoracic empyema in children has been reported. The causative pathogen is often unknown as pleural fluid is frequently sterile at the time of culture. The role of unusual organisms is unclear. Aims: (1) To compare the detection of organisms in pleural fluid from children with empyema using a molecular technique (16S rDNA polymerase chain reaction (PCR)) and bacterial culture. (2) To compare the concordance of organisms identified using the two techniques and the influence of prior antibiotic treatment on positive detection rate. Methods: Pleural fluid from children admitted with empyema between January 2000 and February 2002 was cultured and additionally analysed using broad range 16S rDNA PCR. Results: Pleural fluid was cultured from 32 patients, aged 1 month–16 years. Median duration of previous antibiotic therapy was 8 days (range 1–42 days). Six samples were culture positive and 22 were PCR positive. A causal organism was detected by PCR alone, after considering results from the local hospital, in 14 patients. There was complete concordance in organisms cultured and detected by PCR. Additional organisms detected by PCR were predominantly S pneumoniae, S pyogenes, and anaerobes. Conclusions: Analysis of pleural fluid by broad range 16S rDNA PCR in addition to culture, increases organism identification in empyema.     

Community-acquired methicillin-resistant Staphylococcus aureus colonization in healthy children attending an outpatient pediatric clinic.

BACKGROUND: We previously showed that children attending an inner city pediatric emergency department were sometimes asymptomatically colonized with clindamycin-susceptible community-acquired methicillin-resistant Staphylococcus aureus (MRSA) and borderline methicillin-resistant S. aureus (BRSA) as well. We wished to ascertain whether healthy children attending an outpatient clinic were colonized with these organisms. Therefore to estimate the prevalence of community-acquired MRSA and BRSA nasal colonization in a well child population, we cultured children attending an inner city pediatric outpatient clinic. STUDY DESIGN: This was a prospective cross-sectional study conducted from January to August, 1999, at a primary care outpatient facility at the University of Chicago. The target population was 500 healthy children < or = 16 years of age who attended this facility to receive well child care. RESULTS: One hundred twenty-two (24.4%) children were colonized with S. aureus. Three of the 122 (2.5%) S. aureus isolates were MRSA; they came from children who lacked predisposing risk factors and were susceptible to clindamycin, gentamicin, trimethoprim-sulfamethoxazole, rifampin and ciprofloxacin. Two (1.6%) additional S. aureus isolates were BRSA; both children had predisposing risk factors for MRSA colonization. The mecA gene was present in the 3 MRSA isolates and absent in both BRSA isolates. CONCLUSIONS: These data document that a reservoir of asymptomatic MRSA colonization exists among healthy children who lack traditional risk factors for MRSA infections.     

细菌16S rRNA基因荧光定量PCR诊断新生儿败血症

目的探讨新生儿败血症的快速可靠诊断方法,以提高临床检测细菌的速度及准确性。方法以细菌16SrRNA基因为靶序列,设计通用引物及TaqMan探针,建立细菌16SrRNA基因实时荧光定量PCR反应体系;选择临床引起新生儿败血症的常见菌如金黄色葡萄球菌、表皮葡萄球菌和大肠埃希菌等进行浓度梯度实验;对临床疑为新生儿败血症的830例感染性疾病的新生儿抽取静脉血分别做细菌16SrRNA基因荧光定量PCR和血培养检测。结果临床常见分离株金黄色葡萄球菌、表皮葡萄球菌、大肠埃希菌荧光定量PCR检测结果均为阳性;巨细胞病毒、EB病毒、乙肝病毒,新型隐球菌及白色念珠菌,人基因组DNA及空白对照均为阴性。细菌荧光定量PCR最低能检测到3个细菌,其荧光定量CT值为37．90;对临床疑为感染性疾病的830例患儿标本中,荧光定量PCR检测血标本阳性率5．18％（43／830）,血培养阳性率2．41％（20／830）,前者明显高于后者,差异具有统计学意义,P〈0．01,30例非感染性疾病患儿血标本荧光定量PCR检测及细菌培养均为阴性。若以血培养作为对照,荧光定量PCR方法的诊断敏感性为100％,特异性为97．16％,正确诊断指数为0．972。结论建立了细菌16SrRNA基因荧光定量PCR诊断新生儿败血症方法。其检测快速、简便,为新生儿败血症提供早期、敏感的病原学诊断依据。     

Outbreak of Kingella kingae skeletal system infections in children in daycare

OBJECTIVE: The objective of this study is to describe the investigation of an outbreak of one culture-proven and two presumptive cases of Kingella kingae osteomyelitis detected within a 15-day period in a daycare center in Israel. METHODS: Surveillance pharyngeal cultures were obtained from all attendees at the index daycare center and from two neighboring facilities. Combined amoxicillin-rifampin prophylaxis was administered to all children aged 6 to 30 months living in the community. K. kingae isolates were typed by pulsed field gel electrophoresis, random amplified polymorphic DNA analysis and sequencing of the rRNA genes. RESULTS: Surveillance cultures showed that four of 11 attendees at the index facility as well as five of 12 and one of 15 attendees at neighboring daycare centers carried K. kingae. Typing of isolates showed that the isolate from a child with osteomyelitis was identical to all other isolates from the same daycare center and different from organisms derived from the other facilities. Administration of prophylactic antibiotics resulted in partial eradication of the organism. CONCLUSIONS: Dissemination of K. kingae in a susceptible pediatric population may result in an outbreak of invasive disease. Our experience suggests the need for increased alertness for clusters of K. kingae infections among children in daycare.     

Etiology of septic arthritis in children: an update for the 1990s

OBJECTIVE: To establish the etiology of septic arthritis in children after implementation of HIB immunization guidelines. METHODS: A retrospective review of all charts with a discharge diagnosis of septic arthritis (ICD-9: 711) from January 1991 to December 1996 at St. Louis Children's Hospital was conducted. RESULTS: Sixty-four patients (male = 58%) were identified, whose median age was 6.0 years. Twenty-one children (33%) were misdiagnosed on initial presentation. An organism was isolated in 38 (59%) of cases. The predominant organisms were Staphylococcus aureus (10 isolates), Group A Streptococcus (4), Enterobacter species (4), Kingella kingae (3), Neisseria meningitides (3), Streptococcus pneumoniae (2), Neisseria gonorrhoeae (2), Candida (2), Staphylococcus epidermidis (2). The only isolate of Haemophilus influenzae type B was in 1992 in an unimmunized 14 month old. CONCLUSIONS: These data confirm Staphylococcus aureus as a frequent pathogen and suggest that H influenzae type B is no longer the predominant isolate in young children with septic arthritis. In addition, early septic arthritis in children is frequently misdiagnosed on initial evaluation.     

Kingella kingae osteoarticular infections in children. A report of a series of eight new cases]

Kingella kingae is a Gram-negative bacillus which belongs to the Neisseriaceae family. Its involvement in osteoarticular infections is relatively recent. METHODS AND RESULTS: We report eight cases of Kingella kingae osteoarticular infections that have been diagnosed at the paediatric surgical centre of Rouen University Hospital since October 1995. Six boys and two girls (mean age: 30.6 months) presented with osteomyelitis in six cases and arthritis in two. Only 75% of patients had a fever at time of diagnosis. The biological findings were slightly modified. All samples were obtained from blood, bone or joint fluid. These samples were systematically inoculated into a blood culture tube. Positive Kingella kingae culture was achieved in seven local samples and in one blood culture. All children received two antibiotics via intravenous injection while waiting for the bacteriologic results. Later, the antibiotic treatment (amoxycillin) was given per os. The mean duration of treatment was 33 days. Patients were given intravenous treatment for a period of only ten days. Six patients were followed up for a period of more than 18 months and outcome was always uneventful. DISCUSSION: Kingella kingae is usually present in the nasopharyngeal mucosa and spreads in the blood due to various infections. Different types of Kingella kingae infection have been reported with a large frequency of osteoarticular infection. CONCLUSION: This type of infection does not present any unusual characteristics as compared to other osteoarticular infections. Because of its antibiotic sensitivity treatment duration could be reduced. Kingella kingae is a fragile microbe and its culture is often difficult; therefore, it is important to use blood culture tubes to inoculate joint fluid and bone samples.     

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目的调查温州育英儿童医院小儿下呼吸道感染的病原菌及其耐药性。方法对2003-01—2004-12温州医学院附属育英儿童医院呼吸病区1763例下呼吸道感染患儿的痰液标本经分离培养,做菌株鉴定和药敏试验。结果共分离培养出病原菌715株,总阳性率为40·6%。其中革兰阴性菌448株,占62·7%;革兰阳性菌148株,占20·7%;真菌119株,占16·6%。革兰阴性菌以肺炎克雷伯菌、大肠埃希菌、铜绿假单胞菌和鲍曼不动杆菌为主。肺炎克雷伯菌和大肠埃希菌产超广谱β-内酰胺酶(ESBLs)的百分率分别为49·3%和46·5%,较敏感的抗生素为亚胺培南、丁胺卡那霉素、环丙沙星、哌拉西林/他唑巴坦和头孢哌酮/舒巴坦;除铜绿假单胞菌对复方新诺明的耐药率为100%外,铜绿假单胞菌和鲍曼不动杆菌对各种抗生素的耐药性均较低。革兰阳性菌中以肺炎链球菌和金黄色葡萄球菌为主。肺炎链球菌对青霉素的耐药率达到71·1%,对环丙沙星和万古霉素敏感,耐药率为0。金黄色葡萄球菌中耐甲氧西林金葡菌(MRSA)占18·0%(9/50),对环丙沙星、左旋氧氟沙星和万古霉素敏感。结论温州地区小儿下呼吸道感染的病原菌以革兰阴性菌为主,肺炎克雷伯菌、大肠埃希菌、铜绿假单胞菌、鲍曼不动杆菌、肺炎链球菌、金黄色葡萄球菌为主要病原菌。对抗生素的耐药性较强,临床上应注意对这些菌株的检测,积极防治。     

Frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction

BACKGROUND: Respiratory viruses are the main cause of acute respiratory tract infection (ARI) in children. Real-time polymerase chain reaction (PCR) technology is highly practicable for the rapid detection of viral pathogens. The simultaneous detection of a broad spectrum of viruses enables the diagnosis and evaluation of viral coinfection in ARI. METHODS: A 1-step real-time PCR was developed for the detection of 12 respiratory viruses (10 RNA and 2 DNA viruses) in clinical samples. Clinical samples from 254 children admitted to the Departments of Pediatrics with ARI during a 10-month period were tested. RESULTS: Respiratory syncytial virus (RSV) was the most frequently detected pathogen in 112 samples (44.1%), followed by human bocavirus (hBoV) in 49 (19.3%), and rhinovirus in 17 samples (6.7%). Viral coinfection was detected in 41 (16.1%) samples with RSV and hBoV being the most dominating combination (27 cases, 10.6%). Viral coinfection was found in 10 cases (17%) of children with bronchitis (n = 58) and in 7 cases (23%) of bronchiolitis (n = 30). In patients with pneumonia (n = 51), 17 cases (33%) were positive for 2 or more viral pathogens. CONCLUSIONS: Simultaneous testing of respiratory viruses by real-time PCR is a suitable tool for the detection of viral coinfections. In children hospitalized because of respiratory infection viral coinfection is frequently detected with RSV and hBoV being a common combination.     

Real-time PCR of the 16S-rRNA gene in the diagnosis of neonatal bacteraemia

Abstract Objective: To evaluate a real-time PCR assay for the diagnosis of neonatal bacteraemia. Patients and methods: Two hundred ninety-five plasma samples from 288 newborns with suspected neonatal sepsis were collected prospectively for the purpose of polymerase chain reaction (PCR)-based bacterial detection. A real-time PCR targeting the bacterial gene for 16S-rRNA gene combined with four specific probes designed to detect Gram-negative bacteria, Staphylococcus aureus and coagulase-negative staphylococci (CoNS) was developed. All samples positive in the universal PCR were further sequenced for bacterial identification. Results: When applied to a material from 50 patients with positive blood culture and 245 patients with negative blood culture, the universal PCR showed a sensitivity of 42% (28-57), a specificity of 95% (92-97), a positive predictive value of 64% (45-80), and a negative predictive value of 89% (84-92) (95% confidence intervals in brackets). Conclusion: A new real-time PCR technique was for the first time applied to a well-defined prospectively and consecutively enrolled material of newborns with suspected sepsis, combining the benefits of real-time PCR with specific probes and sequencing. The method managed to detect bacteraemia with high specificity even though the sensitivity was low. Factors causing the low sensitivity are identified and further strategies to develop the method are described.     

Joint and bone infections due to anaerobic bacteria in children

The current review describes the microbiology, diagnosis and management of septic arthritis and osteomyelitis due to anaerobic bacteria in children. Staphylococcus aureus, Haemophilus influenzae type-b, and Group A streptococcus, Streptococcus pneumoniae, Kingela kingae, Neisseria meningiditis and Salmonella spp are the predominant aerobic bacteria that cause arthritis in children. Gonococcal arthritis can occur in sexually active adolescents. The predominant aerobes causing osteomyelitis in children are S. aureus, H. influenzae type-b, Gram-negative enteric bacteria, beta-hemolytic streptococci, S. pneumoniae, K. kingae, Bartonella henselae and Borrelia burgdorferi. Anaerobes have rarely been reported as a cause of these infections in children. The main anaerobes in arthritis include anaerobic Gram negative bacilli including Bacteroides fragilis group, Fusobacterium spp., Clostridium spp. and Peptostreptococcus spp. Most of the cases of anaerobic arthritis, in contrast to anaerobic osteomyelitis, involved a single isolate. Most of the cases of anaerobic arthritis are secondary to hematogenous spread. Many patients with osteomyelitis due to anaerobic bacteria have evidence of anaerobic infection elsewhere in the body, which is the source of the organisms involved in osteomyelitis. Treatment of arthritis and osteomyelitis involving anaerobic bacteria includes symptomatic therapy, immobilization in some cases, adequate drainage of purulent material and antibiotic therapy effective to these organisms.     

Prospective study of 125 cases of Staphylococcus aureus bacteremia in children in New Zealand.

BACKGROUND: Staphylococcus aureus bacteremia is a common complication of S. aureus infection. There are few pediatric studies defining the incidence and associated morbidity and mortality of S. aureus bacteremia and no such New Zealand studies. We conducted a prospective study of S. aureus bacteremia in children in New Zealand. METHODS: From July 1, 1996 to December 31, 1998, we included all children < 16 years of age with S. aureus bacteremia in Auckland and Christchurch. Relevant information regarding patient demographics, clinical course and outcome and laboratory results was recorded. RESULTS: One hundred twenty-five cases of true S. aureus bacteremia were identified. There were 4 deaths within 30 days of the onset of bacteremia. Fourteen (11%) of the children were < 1 month of age. Maori children (relative risk, 2.0; 95% confidence interval, 1.3 to 3.2) were twice as likely and Pacific Island children (relative risk, 2.5; 95% confidence interval, 1.6 to 3.8) 2.5 times as likely as white children to acquire S. aureus bacteremia. The peak incidence of S. aureus bacteremia was observed in Pacific Island children < 1 year of age (105 cases/100,000 children/year). Twenty-seven percent of cases were related to intravenous catheters. Seventy percent of cases were community-acquired. Ninety-eight percent of non-catheter-related cases in children > 1 month of age were community-acquired. There was a low rate of methicillin resistance (6%). CONCLUSIONS: S. aureus bacteremia is largely community-acquired in children in New Zealand and is more common in Pacific Island and Maori populations. Although there is a low associated mortality, a significant number are potentially preventable cases secondary to intravenous catheters.     

Rapid diagnosis of bacterial meningitis by using multiplex PCR and real time PCR

BACKGROUND: The purpose of the present study was to improve a method for a rapid identification of bacteria in bacterial meningitis by using multiplex polymerase chain reaction (PCR). METHODS: Ten species of bacteria which cause meningitis in children were investigated, and cerebrospinal fluid from patients with purulent meningitis was studied. The ribosomal RNA genes of bacteria are essential, and are highly conserved in the bacterial kingdoms with consensus region. The 23S rRNA region shows a larger variation among species than in the 16S rRNA region. The authors set primers in the universal region and specific region of 23S rRNA, then amplified these regions by multiplex PCR and real-time PCR. RESULTS: All species of bacteria showed one band by PCR using universal primer. Haemophilus influenzae and Streptococcus pneumoniae showed two bands by multiplex PCR using a combination of universal primers and specific primers. The authors detected H. influenzae within 15 min by using real-time PCR. CONCLUSION: It was possible to identify clinically significant bacterial species in cerebrospinal fluid by multiplex PCR, and to identify H. influenzae by real-time PCR within a short period.     

Comparison of 16S rDNA-PCR Amplification and Culture of Cerebrospinal Fluid for Diagnosis of Bacterial Meningitis

Objective

Early and accurate diagnosis of bacterial meningitis is of critical concern. Optimum and rapid laboratory facilities are not routinely available for detecting the etiologic agents of meningitis. The objective of this study was to compare polymerase chain reaction (PCR) assay with culture for detection of bacteria in central nervous system (CNS) samples from patients suspected to have meningitis.

Methods

One-hundred cerebrospinal fluid (CSF) samples were obtained and divided into two parts. One part of samples was used for standard bacterial culture and gram staining. The remaining was used for DNA extraction. PCR assay was performed with universal primers for 16S rDNA gene of bacteria. Performance characteristics of the test were determined.

Findings

The PCR method was able to detect bacteria in all 36 culture-positive and in 38 of 64 culture-negative cases showing sensitivity and specificity of 100% and 40.6% respectively. Positive predictive value was 48.6% and negative predictive value 100%, however, Kappa coefficient showed the correlation of the 2 methods to be at 0.33.

Conclusion

There are advantages and disadvantages in performance characteristics of the conventional CSF culture and universal CSF 16S rDNA PCR. Therefore, it is recommended to use both methods in clinical practice, particularly in suspicious contaminated samples, with presumable presence of fastidious or slow growing bacteria because of antibiotic consumption.