Richard F. de Boer

Prospective study of use of PCR amplification and sequencing of 16S ribosomal DNA from cerebrospinal fluid for diagnosis of bacterial meningitis in a clinical setting.

ABSTRACT We have evaluated the use of a broad-range PCR aimed at the 16S rRNA gene in detecting bacterial meningitis in a clinical setting. To achieve a uniform DNA extraction procedure for both gram-positive and gram-negative organisms, a combination of physical disruption (bead beating) and a silica-guanidiniumthiocyanate procedure was used for nucleic acid preparation. To diminish the risk of contamination as much as possible, we chose to amplify almost the entire 16S rRNA gene. The analytical sensitivity of the assay was approximately 1 × 102 to 2 × 102 CFU/ml of cerebrospinal fluid (CSF) for both gram-negative and gram-positive bacteria. In a prospective study of 227 CSF samples, broad-range PCR proved to be superior to conventional methods in detecting bacterial meningitis when antimicrobial therapy had already started. Overall, our assay showed a sensitivity of 86%, a specificity of 97%, a positive predictive value of 80%, and a negative predictive value of 98% compared to culture. We are currently adapting the standard procedures in our laboratory for detecting bacterial meningitis; broad-range 16S ribosomal DNA PCR detection is indicated when antimicrobial therapy has already started at time of lumbar puncture or when cultures remain negative, although the suspicion of bacterial meningitis remains.

Improved Detection of Five Major Gastrointestinal Pathogens by Use of a Molecular Screening Approach

ABSTRACT The detection of bacterial and parasitic gastrointestinal pathogens through culture and microscopy is laborious and time-consuming. We evaluated a molecular screening approach (MSA) for the detection of five major enteric pathogens: Salmonella enterica, Campylobacter jejuni, Giardia lamblia, Shiga toxin-producing Escherichia coli (STEC), and Shigella spp./enteroinvasive E. coli (EIEC), for use in the daily practice of a clinical microbiology laboratory. The MSA consists of prescreening of stool specimens with two real-time multiplex PCR (mPCR) assays, which give results within a single working day, followed by guided culture/microscopy of the positive or mPCR-inhibited samples. In the present 2-year overview, 28,185 stool specimens were included. The MSA was applied to 13,974 stool samples (49.6%), whereas 14,211 samples were tested by conventional methods only (50.4%). The MSA significantly increased the total detection rate compared to that of conventional methods (19.2% versus 6.4%). The detection of all included pathogens, with the exception of S. enterica, significantly improved. MSA detection frequencies were as follows: C. jejuni, 8.1%; G. lamblia, 4.7%; S. enterica, 3.0%; STEC, 1.9%; and Shigella spp./EIEC, 1.4%. The guided culture/microscopy was positive in 76.8%, 58.1%, 88.9%, 16.8%, and 18.1% of mPCR-positive specimens, respectively. Of all mPCRs, only 1.8% was inhibited. Other findings were that detection of mixed infections was increased (0.9% versus 0.02%) and threshold cycle (CT ) values for MSA guided culture/microscopy-positive samples were significantly lower than those for guided culture/microscopy-negative samples. In conclusion, an MSA for detection of gastrointestinal pathogens resulted in markedly improved detection rates and a substantial decrease in time to reporting of (preliminary) results.

Streptococcus pneumoniae DNA Load in Blood as a Marker of Infection in Patients with Community-Acquired Pneumonia

ABSTRACT Direct detection of Streptococcus pneumoniae DNA in blood adds to culture results in the etiological diagnosis of patients with community-acquired pneumonia (CAP). Quantification of the amount of DNA, the bacterial DNA load (BDL), provides a measurement of DNAemia that may increase the understanding of the clinical relevance of S. pneumoniae DNA in blood. We evaluated the S. pneumoniae BDL as a diagnostic tool in adult patients with CAP. The BDL was determined in whole-blood samples collected simultaneously with blood for culture from 45 adult patients with CAP. After DNA extraction, S. pneumoniae DNA was detected with specific real-time PCR amplification, and the BDL was calculated with a standard curve. PCR and microbiological results were compared, and the BDL was related to clinical and laboratory parameters. S. pneumoniae DNA was detected in 10/13 patients with positive blood cultures and in 67% of patients with microbiologically confirmed pneumococcal pneumonia. The positive predictive values of the receiver operating characteristic curves for the BDLs for pneumococcal infection (100%) and pneumococcal bacteremia (69%) were higher than those for the level of C-reactive protein (CRP; 43% and 23%, respectively) and the white blood cell count (WBC; 42% and 35%, respectively); the negative predictive values of these three parameters were in the same range (±90 and ±97%, respectively). The BDL was higher in patients presenting with systemic inflammatory response syndrome and in patients with bacteremia. Positive correlations were observed for the BDL with WBC, CRP level, and length of stay. We conclude that the BDL supports the diagnosis of S. pneumoniae infection in patients with CAP and provides a putative marker of the severity of disease.

Premature and accelerated aging: HIV or HAART?

Highly active antiretroviral therapy (HAART) has significantly increased life expectancy of the human immunodeficiency virus (HIV)-positive population. Nevertheless, the average lifespan of HIV-patients remains shorter compared to uninfected individuals. Immunosenescence, a current explanation for this difference invokes heavily on viral stimulus despite HAART efficiency in viral suppression. We propose here that the premature and accelerated aging of HIV-patients can also be caused by adverse effects of antiretroviral drugs, specifically those that affect the mitochondria. The nucleoside reverse transcriptase inhibitor (NRTI) antiretroviral drug class for instance, is known to cause depletion of mitochondrial DNA via inhibition of the mitochondrial specific DNA polymerase-γ. Besides NRTIs, other antiretroviral drug classes such as protease inhibitors also cause severe mitochondrial damage by increasing oxidative stress and diminishing mitochondrial function. We also discuss important areas for future research and argue in favor of the use of Caenorhabditis elegans as a novel model system for studying these effects.

Improved detection of microbial DNA after bead-beating before DNA isolation

Detection of microbial DNA in clinical samples by real-time PCR requires a universal extraction method with equally efficient lysis of cell walls of all possible microorganisms. We demonstrated that physical disruption by bead-beating with 0.1mm beads in combination with MagNA Pure DNA III extraction enhances microbial lysis of diverse Gram-positive microorganisms and may be used to optimize DNA extraction protocols in routine clinical diagnostics.

Reduced PCR Sensitivity Due to Impaired DNA Recovery with the MagNA Pure LC Total Nucleic Acid Isolation Kit

ABSTRACT The increasing demand for molecular diagnostics in clinical microbiology laboratories necessitates automated sample processing. In the present study, we evaluated the performance of the MagNA Pure LC total nucleic acid isolation kit (M extraction) in comparison with the manual method (Si extraction) according to Boom et al. (R. Boom, C. J. A. Sol, M. M. M. Salimans, C. L. Jansen, P. M. Wertheim-van Dillen, and J. van der Noordaa, J. Clin. Microbiol. 28:495-503, 1990) for the detection of viral DNA by competitive quantitative PCR. Reconstruction experiments with HindIII-digested phage lambda DNA and HaeIII-digested φX174 DNA showed that the recovery of DNA from phosphate-buffered saline, cerebrospinal fluid, EDTA-anticoagulated plasma, and EDTA-anticoagulated whole blood by M extraction is, on average, 6.6-fold lower compared to Si extraction. PCR signals of spiked PCR control DNAs for Epstein-Barr virus and varicella-zoster virus were also between 1.9- and 14.2-fold lower after M extraction compared to Si extraction, also suggesting impaired DNA recovery. M extraction of spiked cytomegalovirus strain AD 169 in whole blood showed a 5- to 10-fold reduction in PCR sensitivity compared to Si extraction. This reduction of PCR sensitivity was also observed when clinical whole blood samples were processed by M extraction. Before implementing M extraction, the clinical consequences of the reduced recovery should first be considered, especially when maximal sensitivity is required.

High Detection Rates of Enteropathogens in Asymptomatic Children Attending Day Care

Background Gastroenteritis morbidity is high among children under the age of four, especially amongst those who attend day care. Objective To determine the prevalence of a range of enteropathogens in the intestinal flora of children attending day care and to relate their occurrence with characteristics of the sampled child and the sampling season. Methods We performed three years of enteropathogen surveillance in a network of 29 child day care centers in the Netherlands. The centers were instructed to take one fecal sample from ten randomly chosen children each month, regardless of gastrointestinal symptoms at time of sampling. All samples were analyzed for the molecular detection of 16 enteropathogenic bacteria, parasites and viruses by real-time multiplex PCR. Results Enteropathogens were detected in 78.0% of the 5197 fecal samples. Of the total, 95.4% of samples were obtained from children who had no gastroenteritis symptoms at time of sampling. Bacterial enteropathogens were detected most often (most prevalent EPEC, 19.9%), followed by parasitic enteropathogens (most prevalent: D. fragilis, 22.1%) and viral enteropathogens (most prevalent: norovirus, 9.5%). 4.6% of samples related to children that experienced symptoms of gastroenteritis at time of sampling. Only rotavirus and norovirus were significantly associated with gastroenteritis among day care attendees. Conclusions Our study indicates that asymptomatic infections with enteropathogens in day care attendees are not a rare event and that gastroenteritis caused by infections with these enteropathogens is only one expression of their presence.

Detection of Campylobacter Species and Arcobacter butzleri in Stool Samples by Use of Real-Time Multiplex PCR

ABSTRACT The presence of Campylobacter (or Campylobacter-like) species in stools from patients suspected of infectious gastroenteritis (n = 493) was investigated using real-time PCR for detection of Arcobacter butzleri (hsp60 gene), Campylobacter coli (ceuE gene), Campylobacter jejuni (mapA), five acknowledged pathogenic Campylobacter spp. (C16S_Lund assay), and the Campylobacter genus (C16S_LvI assay). In total, 71.4% of the samples were positive for Campylobacter DNA (n = 352) by a Campylobacter genus-specific (C16S_LvI) assay. A total of 23 samples (4.7%) were positive in the C16S_Lund assay, used for detection of C. jejuni, C. coli, C. lari, C. upsaliensis, and C. hyointestinalis. Subsequent identification of these samples yielded detection frequencies (DF) of 4.1% (C. jejuni), 0.4% (C. coli), and 0.4% (C. upsaliensis). The DF of A. butzleri was 0.4%. Interestingly, sequencing of a subgroup (n = 46) of C16S_LvI PCR-positive samples resulted in a considerable number of Campylobacter concisus-positive samples (n = 20). PCR-positive findings with the C16S_Lund and C. jejuni/C. coli-specific assays were associated with more serious clinical symptoms (diarrhea and blood). Threshold cycle (CT ) values of C. jejuni/C. coli PCR-positive samples were comparable to those of the C16S_Lund PCR (P = 0.21). CT values for both assays were significantly lower than those of the C16S_LvI assay (P < 0.001 and P < 0.00001, respectively). In conclusion, this study demonstrated that in combination, the C. jejuni/C coli-specific assays and the C16S_Lund assay are both useful for routine screening purposes. Furthermore, the DF of the emerging pathogen C. concisus was at least similar to the DF of C. jejuni.

Tin tetrachloride-induced π-cyclizations of glycine cation equivalents to substituted pipecolic acid derivatives

Abstract Cationic π-cyclization reactions of N-(3-alkenyl)-N-(methoxycarbonyl)acetoxyglycine esters induced by tin tetrachloride in dichloromethane are described. Reactions started and quenched with water at-78 °C mainly yield cis-4-hydroxypipecolic esters, whereas reactions quenched after warm-up to room temperature provide trans-4-chloropipecolic esters as major products. A mechanistic scheme is advanced which adequately explains these results. The essentials are a rapid cationic aza-Cope equilibrium of the incipient iminium cation, and participation of the ester moiety through formation of a relatively stable bicyclic dioxycarbenium cadon as pivotal intermediate.

Reductive cyclization of carbon-centered glycine radicals; a novel synthetic route to cyclic α-amino acids

Reductive cyclizations (tributylin hydride, AIBN) of several α-(penthylthio)glycine derivatives with a 30alkenyl substituent at nitrogen are reported. These reactions proceed via 2-aza-5-alken-1-yl radicals as intermediates which bear electron-withdrawing carbonyl substituents at the radical center and at nitrogen. Such radicals can be considered as relatively stable captodative radicals, but are reactive enough for olefin cyclization. The main products usually arise from 5-exo cyclization and are structurally interesting analogues of proline. Varying amounts of pipecolic acid analogues via 6-endo cyclization are also obtained in some cases. A similar cyclization of an acetylene is successful, whereas a nitrile fails to cyclize.