Amel Kechrid

Target Gene Sequencing To Characterize the Penicillin G Susceptibility of Neisseria meningitidis

ABSTRACT Clinical isolates of Neisseria meningitidis with reduced susceptibility to penicillin G (intermediate isolates, PenI) harbor alterations in the penA gene encoding the penicillin binding protein 2 (PBP2). A 402-bp DNA fragment in the 3′ half of penA was sequenced from a collection of 1,670 meningococcal clinical isolates from 22 countries that spanned 60 years. Phenotyping, genotyping, and the determination of MICs of penicillin G were also performed. A total of 139 different penA alleles were detected with 38 alleles that were highly related, clustered together in maximum-likelihood analysis and corresponded to the penicillin G-susceptible isolates. The remaining 101 penA alleles were highly diverse, corresponded to different genotypes or phenotypes, and accounted for 38% of isolates, but no clonal expansion was detected. Analysis of the altered alleles that were represented by at least five isolates showed high correlation with the PenI phenotype. The deduced amino acid sequence of the corresponding PBP2 comprised five amino acid residues that were always altered. This correlation was not complete for rare alleles, suggesting that other mechanisms may also be involved in conferring reduced susceptibility to penicillin. Evidence of mosaic structures through events of interspecies recombination was also detected in altered alleles. A new website was created based on the data from this work (http://neisseria.org/nm/typing/penA ). These data argue for the use of penA sequencing to identify isolates with reduced susceptibility to penicillin G and as a tool to improve typing of meningococcal isolates, as well as to analyze DNA exchange among Neisseria species.

The diagnosis of pertussis: which method to choose?

Despite the introduction of routine vaccination against pertussis for more than a half century, leading to a drastic decline in the number of reported cases, pertussis continues to be an important respiratory disease afflicting unvaccinated infants and previously vaccinated children as well as adults in whom immunity has waned. The diagnosis of pertussis is challenging and accurate laboratory identification of Bordetella infections remains problematic. Common laboratory diagnostic methods used for pertussis diagnosis include culture, direct-fluorescent-antibody testing (DFA), serology and polymerase chain reaction (PCR). Culture of Bordetella pertussis is highly specific but fastidious and has limited sensitivity. DFA provides a much more rapid result, but has the disadvantage of poor sensitivity and specificity. Serology is not useful in infants. In older persons, it is hampered by the limitations of paired sera and it provides mainly a retrospective diagnosis. Such limitations of conventional diagnosis testing have led to the development of PCR assays. Notwithstanding its lack of standardization, PCR has been found to be more sensitive and more specific than other methods. In this report, we aimed to review current knowledge about the available diagnostic methods and tests that accurately diagnose pertussis.

Dual infection with Bordetella pertussis and Mycoplasma pneumoniae in three infants: case reports

Studying pertussis-like respiratory infections, we report the cases of three infants with evidence of both Bordetella pertussis and Mycoplasma pneumoniae. Bordetella infection was identified by the real-time polymerase chain reaction (RT-PCR) of nasopharyngeal specimens. Neither B. pertussis nor B. parapertussis were recovered on the culture of nasopharyngeal aspirates (NPAs) from any subjects. M. pneumoniae etiology was diagnosed by culture and RT-PCR. The evolution was fatal for all of the subjects. We conclude that, among patients with Bordetella infection, co-infection with another respiratory pathogen is often probable, and these mixed infections might cause a more severe form of illness, sometimes leading to death.

Predictors of Mortality in Mechanically Ventilated Critical Pertussis in a low Income Country

Background Critical pertussis is characterized by severe respiratory failure, important leukocytosis, pulmonary hypertension, septic shock and encephalopathy. Aim To describe the clinical course of critical pertussis, and identify predictors of death at the time of presentation for medical care. Methodology Retrospective study conducted in children’s hospital Tunisian PICU between 01 January and 31 October 2013. Patients with critical pertussis confirmed by RT-PCR and requiring mechanical ventilation were included. Predictors of death were studied. Results A total of 17 patients was studied. Median age was 50 days. Mortality was 23%. Predictors risk of mortality were : high PRISM score (Pediatric Risk of Mortality Score) (p=0,007), shock (p=0,002), tachycardia (p=0,005), seizures (p=0,006), altered mental status (p=0,006), elevated WBC count (p=0,003) and hemodynamic support (p=0022). However, the difference did not reach statistical significance in comorbidity, pneumoniae, high pulmonary hypertension or exchange transfusion. Concomitant viral or bacterial co-infection was not related to poor outcome. Conclusion Young infants are at high risk to have critical pertussis. Despite advances in life support and the treatment of organ failure in childhood critical illness, critical pertussis remains difficult to treat.

Méningite bactérienne iatrogène après ponction lombaire de diagnostic: à propos de trois cas observés à l’hôpital d’enfants de Tunis

We have collected cases of iatrogenic meningitis managed in the Childrens Hospital of Tunis, between January 1998 and December 2006. Clinical information about each patient were collected, all bacterial samples were investigated in the microbiology laboratory of the hospital. Bacterial isolates were identified according to conventional criteria. In the interval under study, we recorded three cases of iatrogenic meningitis after lumbar puncture. Two cases occurred in newborn admitted for suspicion of neonatal infection and one in a 2-month-old infant admitted for exploration of hyperpyretic convulsion. In all patients, the initial cerebrospinal fluid was normal. All patients developed symptoms of acute meningitis within 72 hours after lumbar puncture; the second cerebrospinal fluid was, then, typical for purulent meningitis. The causal agents isolated in the three cases were Klebsiella pneumoniae, Enterobacter cloacae, and Serratia marcescens, all resistant to beta-lactams by extended spectrum beta-lactamase production. The use of quinolones was required in all cases. Different complications were recorded: hydrocephalus and brain abscess in one case, respiratory and hemodynamic failure managed in the intensive care unit in the second, and brain hygroma in the third case. This study shows high morbidity of iatrogenic meningitis. Simple aseptic precautions undertaken before the procedure of lumbar puncture can prevent such cases. The urgent need for increasing the awareness among medical personnel in hospitals of developing countries cannot be overemphasized.

Pneumococcal Osteomyelitis in Children: A Case Report

We describe a case of osteomyelitis, in a 7-month-old boy, caused by S. pneumoniae involving the pelvic bone with a purulent collection in the adductor muscle. Osteomyelitis diagnosis was suspected using ultrasound and CT scan investigations. This showed a periosteal elevation in the Case Study Ben Marzouk et al.; IJMPCR, 3(3): 68-72, 2015; Article no.IJMPCR.2015.038 69 pelvis and an abscess of the adductor muscle. Culture of the perioperative specimens isolated serotype 19A S. pneumoniae with reduced susceptibility to penicillin and macrolides. The patient was treated successfully with amoxicillin-clavulanate during five weeks, associated with gentamicin during the first week.

867 Epidemiology of Bacterial Meningitis in Tunisian Children (2000–2011)

Bacterial meningitis is associated with high mortality and neurological squeal world wide. We reported epidemiological characteristics of laboratory confirmed bacterial meningitis in children during 2000 to 2011 period. We analyzed all laboratory confirmed bacterial meningitis cases. The serotypes and serogroups were determined by slide agglutination. Antibiotic susceptibility was determined by disk diffusion method according to CA-SFM guidelines. Beta-lactamase production was analyzed using cefinase test. MIC of beta-lactams was determined by E-test method (AB BIODISK). During the study period we have collected 486 cases of bacterial meningitis: 157 cases of S. pneumoniae (32.3%), 118 of N. meningitidis (24.3%), and 99 of H. influenzae (20.4%). Most cases (66.5%) occurred in children under 3 years. The most frequent serotype among S. pneumoniae was 14 (27.2%) followed by 23F (9%). The majority of N meningitidis strains belonged to serogroup B (72.9%) and 88.8% of H. influenzae strains to serotype b. Before Hib conjugate vaccine introduction (2000–2002), H. influenzae were the first species responsible for meningitis (40%). During vaccine generalisation (2003–2005) we noted a decrease in Hib meningitis. At the beginning of 2006, Hib vaccination was stopped and we observed an increase of Hib meningitis cases. Antimicrobial susceptibility studies show that 43.6% and 60% respectively of S. pneumoniae and N. meningitidis strains had reduced susceptibility to penicillin. Among H. influenzae 47.3% was beta-lactamase producing. The data presented in this study demonstrate that S. pneumoniae is the most frequent in bacterial meningitis in children and that beta-lactams resistance is frequent in our hospital.

926 Haemophilus Influenzae in Children: Resistance to Six Other BÊTa-Lactams Among Ampicillin-Resistant Strains

Background and Aims Haemophilus influenzae (Hi) is a human pathogen responsible for various infections in both children and adults. We describe in this study the susceptibility patterns and β-lactam resistance mechanisms of 62 ampicillin-resistant H.i strains isolated from children at the children’s hospital of Tunis during 2009 and 2010. Materials and Methods All strains were identified and serotyped using conventional methods. Antimicrobial susceptibility was determined by E-test. The antibiotics tested were amoxicillin, amoxicillin-clavulanate, cefixim, cefuroxim, cefotaxim, cefpodoxim and imipenem. The β-lactamase production was performed using the nitrocefin test. We determined the resistance genes (bla TEM-1, bla ROB-1 and ftsI) by PCR. Results Isolates were identified as non capsulated and were classified into 3 groups according to their β-lactam resistance mechanisms: β-lactamase positive ampicillin-resistant (BLPAR: 50%); β-lactamase negative ampicillin-resistant (BLNAR: 40.32%) and β-lactamase positive amoxicillin-clavulanate-resistant (BLPACR: 9.68%). All strains showed high amoxicillin, amoxicillin-clavulanate, cefuroxim and imipenem MICs. Among these, the less active one was imipenem with MIC50 >32mg/l in all strains. The highest MICs of cefuroxim were in BLPACR strains (2–4mg/l). MICs ranges of this antibiotic were 0.5–6 mg/l in BLNAR and 0.125–4 mg/l in BLPAR. Cefotaxim, cefixim and cefpodoxim were the most active agents tested against our strains. Conclusion This study indicates that many β-lactams are ineffective among some Hi strains. So, it’s important to have an appropriate usage of antibiotics to stop these phenomena. We must make other investigations to know if these strains belonged to the same clone or if it’s a question of an outbreak in our hospital.

479 Biological Diagnosis of BordetellaInfections And Determination of their Epidemiological Characteristics in Tunisian Infants < 1 Year of Age

Background and Aims Bordetella pertussis and B. parapertussis are the causative agents of whooping cough, a re-emerging infectious disease in spite of reasonable vaccination coverage. Specific diagnostic tools were applied for the first time in a Tunisian prospective study in order to get an estimation of the prevalence of Bordetella infections, and to evaluate their use to determine the epidemiological characteristics of these infections in Tunisian infants. Methods Between 2007 and 2011, a total of 626 samples from 599 infants aged < 1 year with and without pertussoid cough were investigated for the presence of B. pertussis/parapertussis using culture and real-time PCR (RT-PCR). When possible, patients’ household contacts provided nasopharyngeal aspirates (NPAs) for RT-PCR detection of B. pertussis/parapertussis or single-serum samples for anti-PT IgG quantification. Results All except 1 NPA were negative by conventional culture whereas PCR gave positive signals for 126 specimens (21%): B. pertussis, B. parapertussis and Bordetella spp. were detected in 82, 6 and 4% of the samples, respectively. The simultaneous presence of B. pertussis and B. parapertussis was noted in 8% of the cases. The prevalence of Bordetella infection was 20%. Most of these cases corresponded to patients younger than 6 months old who received < 3 doses of pertussis vaccine. Among the household contacts enrolled in the study, mothers were the likely source of infection in 4 cases. Conclusions This study showed that pertussis is still prevalent in Tunisia and the disease remains a public health problem affecting not only infants but also adults.

Méningite bactérienne iatrogène après ponction lombaire de diagnostic : à propos de trois cas observés à l'hôpital d'enfants de Tunis Iatrogenic meningitis after diagnosis lumbar puncture: 3 cases reports in the paediatric Children's Hospital of Tunis

We have collected cases of iatrogenic meningitis managed in the Childrens Hospital of Tunis, between January 1998 and December 2006. Clinical information about each patient were collected, all bacterial samples were investigated in the microbiology laboratory of the hospital. Bacterial isolates were identified according to conventional criteria. In the interval under study, we recorded three cases of iatrogenic meningitis after lumbar puncture. Two cases occurred in newborn admitted for suspicion of neonatal infection and one in a 2-month-old infant admitted for exploration of hyperpyretic convulsion. In all patients, the initial cerebrospinal fluid was normal. All patients developed symptoms of acute meningitis within 72 hours after lumbar puncture; the second cerebrospinal fluid was, then, typical for purulent meningitis. The causal agents isolated in the three cases were Klebsiella pneumoniae, Enterobacter cloacae, and Serratia marcescens, all resistant to beta-lactams by extended spectrum beta-lactamase production. The use of quinolones was required in all cases. Different complications were recorded: hydrocephalus and brain abscess in one case, respiratory and hemodynamic failure managed in the intensive care unit in the second, and brain hygroma in the third case. This study shows high morbidity of iatrogenic meningitis. Simple aseptic precautions undertaken before the procedure of lumbar puncture can prevent such cases. The urgent need for increasing the awareness among medical personnel in hospitals of developing countries cannot be overemphasized.