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The determination of herpes simplex virus (HSV) infection using a PCR assay is one of the most commonly requested tests for analysis of cerebrospinal fluid (CSF), although only a very low proportion of results are positive. A previously reported study showed that selecting only those CSF samples with >5 leukocytes/mm 3 or a protein level of >50 mg/dl was adequate for the diagnostic workup. The aim of the present study was to assess the reliability of alternative acceptance criteria based on elevated CSF white blood cell counts (>10 cells/mm 3 ). We analyzed all requests for HSV PCR received between January 2008 and December 2011. CSF samples were accepted for analysis if they had >10 cells/mm 3 or if the sample was from an immunocompromised patient or a child aged <2 years. In order to evaluate our selection criteria, we identified those CSF samples with a leukocyte count of 5 to 10 cells/mm 3 or protein levels of >50 mg/dl in order to test them for HSV type 1 and 2 (HSV-1 and HSV-2) DNA. During the study period, 466 CSF samples were submitted to the microbiology laboratory for HSV PCR. Of these, 268 (57.5%) were rejected, and 198 (42.5%) were tested according to our routine criteria. Of the tested samples, 11 (5.5%) were positive for HSV DNA (7 for HSV-1 and 4 for HSV-2). Of the 268 rejected specimens, 74 met the criteria of >5 cells/mm 3 and/or protein levels of >50 mg/dl. Of these, 70 (94.6%) were available for analysis. None of the samples yielded a positive HSV PCR result. Acceptance criteria based on CSF leukocyte counts, host immune status, and age can help to streamline the application of HSV PCR without reducing sensitivity. Detection of herpes simplex virus (HSV) DNA in cerebrospinal fluid (CSF) using PCR assay has been validated for the diagnosis of central nervous system (CNS) herpes infections. HSV PCR is currently recognized as the reference method (1, 2). This sensitive but expensive test is included in the routine evaluation of many patients with suspected CNS infection, although most of the tests performed yield negative results (3). Therefore, screening for suitable diagnostic samples is necessary.Acceptance criteria based on elevated CSF leukocyte counts (Ͼ5 cells/mm 3 ) and protein levels (Ͼ50 mg/dl) have been proposed as a way to save health care costs without reducing sensitivity (4, 5); however, other acceptance criteria have not been evaluated.Most patients with viral CNS infection have an abnormal CSF leukocyte count, which usually ranges from 10 cells/mm 3 to 200 cells/mm 3 (6). The CSF leukocyte counts reported in previous studies (1, 2, 5-13) are summarized in Table 1. In our institution, we accepted CSF specimens for HSV PCR testing if they had Ͼ10 cells/mm 3 . This cutoff was based both on our experience before the study and on data from the studies cited above. We also accepted specimens collected from immunocompromised patients and children younger than 2 years of age.Clinical laboratories attempt to decrease costs while maintaining the quality of the diagnostic approaches used. A...

A persistent 8-year infection by a Beijing Mycobacterium tuberculosis strain from a previous outbreak after importation from West Africa obliged us to investigate secondary cases. We developed a multiplex PCR method based on whole-genome sequencing to target strain-specific single nucleotide polymorphisms (SNPs). In 1 week, we analyzed 868 isolates stored over 6 years. Only 2 cases (immigrants from Guinea Conakry) harbored the strain, which ruled out transmission-despite opportunitiesand challenged some of the advantages associated with Beijing strains. Extensive application of genotyping strategies to characterize Mycobacterium tuberculosis has enabled us to identify strains that are advantageous because of their virulence, ability to acquire resistance, or efficiency in causing severe outbreaks.Once such strains are identified, their fingerprints can be used as a reference to closely monitor their emergence in populations where they remain undetected (1). However, this approach requires universal population-based systematic fingerprinting programs.Alternatively, strain-specific PCRs can be used to track the emergence of theoretically advantageous strains (2, 3). Wholegenome sequencing (WGS) has simplified the identification of genotypic peculiarities (based on specific single nucleotide polymorphisms [SNPs]) from which we can design strain-specific PCRs (4, 5).We developed an allele-specific oligonucleotide (ASO)-multiplex PCR method based on WGS data to track the presence of a susceptible Beijing strain in Madrid, Spain. This strain was responsible for a severe outbreak on Gran Canaria Island (GCI) in the 1990s, and it was eventually shown to be the cause of around one-quarter of the total number of cases on the island (6). The GCI strain was still found in 2007 and 2008 as shown after the application of a specific PCR (7). In Madrid, we identified a nonadherent case with persistently active infectious tuberculosis (8 years) caused by the Gran Canaria strain (8), which obliged us to fast track potential secondary cases.(The results of this study were partially presented at the 2015 ESM Meeting, Riga, Latvia, 28 June to 1 July 2015.)The persistent case was diagnosed in our institution in 2006. The mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) pattern of this isolate coincided with the pattern described for the GCI strain (7). Using fingerprinting data from 2006 to 2008, we only detected 3 patients, all of whom were linked to the GCI outbreak. Given that the population-based genotyping program running in our area was discontinued at the end of 2008, we were unable to identify potential secondary cases that occurred from 2009 to 2014, when previously exposed cases were more likely to develop the disease. To update the 2009 to 2014 gap, we developed an ASO-PCR method to retrospectively fast track the GCI strain by directly analyzing stored isolates.WGS of 4 isolates from our case was performed as indicated elsewhere (4). We followed standard library preparation protocols. Us...

dMatrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of nontuberculous mycobacterial (NTM) isolates was evaluated in this study. Overall, 125 NTM isolates were analyzed by MALDI-TOF and GenoType CM/AS. Identification by 16S rRNA/hsp65 sequencing was considered the gold standard. Agreements between MALDI-TOF and GenoType CM/AS with the reference method were, respectively, 94.4% and 84.0%. In 17 cases (13.6%), results provided by GenoType and MALDI-TOF were discordant; however, the reference method agreed with MALDI-TOF in 16/17 cases (94.1%; P ‫؍‬ 0.002). Identification of nontuberculous mycobacteria (NTM) by conventional phenotypic tests requires long incubation periods that may take up to 12 weeks. The GenoType Mycobacterium CM/AS (Hain Lifescience GmbH, Nehren, Germany) is a test based on the amplification of a 23S rRNA gene region followed by reverse hybridization with specific probes that allows the identification of 40 of the most common NTM. However, the hybridization step requires high sequence homology, and even point mutations in the target regions may hamper the hybridization step, leading to unreliable results (1). Matrix-assisted laser desorption ionizationtime of flight (MALDI-TOF) has been recently applied to the identification of a wide range of microorganisms (2), mainly clinically significant bacteria and fungi. Experience with MALDI-TOF for the rapid identification of NTM is very limited, mainly because of the number of identified species and because database information is scarce (3-5). In this study, a large collection of NTM clinical isolates and reference strains were analyzed using MALDI-TOF technology in comparison with the GenoType Mycobacterium CM/AS procedure and the reference procedure, 16S rRNA/hsp65 gene sequencing.From January 2010 to September 2014, all the NTM with purportedly clinical significance were grown on Lowenstein-Jensen agar and simultaneously identified by GenoType CM/AS assay (Hain Lifescience, GmbH, Nehren, Germany) and with MALDI-TOF MS technology (Bruker Daltonics, Bremen, Germany). Ten reference isolates were also included in this study as positive controls (Table 1). All the strains were also analyzed by 16S rRNA/ hsp65 sequencing as the reference method in order to resolve possible discrepancies.Genotypic identification. Two gene regions were targeted for NTM identification: the 5= end of the 16S rRNA gene with the universal primers E8F (5=-AGAGTTTGATCCTGGCTCAG-3=) and E533R (5=-TTACCGCGGCTGCTGGCA-3=) (6) and a 439-bp fragment within the hsp65 gene using the primers TB11 (5=-ACCAACGATGGTGTGTCCAT) and TB12 (5=-CTTGTCGA ACCGCATACCCT), as described by Telenti et al. (7). The amplification, purification, and sequencing process was carried out as previously described by . Interpretation of sequencing results was performed considering CLSI recommendations for the genus Mycobacterium (9). Results obtained by 16S rRNA/hsp65 sequencing were considered the final microorganism identification.GenoType CM/AS iden...