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The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly reached pandemic levels. Sufficient testing for SARS-CoV-2 has remained essential for tracking and containing the virus. SARS-CoV-2 testing capabilities are still limited in many countries. Here, we explore the use of conventional RNA purification as an alternative to automated systems for detection of SARS-CoV-2 by RT-qPCR. 87 clinical swab specimens were extracted by conventional phenol-chloroform RNA purification and compared to commercial platforms for RNA extraction and the fully integrated Cobas®6800 diagnostic system. Our results show that the conventional RNA extraction is fully comparable to modern automated systems regarding analytical sensitivity and specificity with respect to detection of SARS-CoV-2 as evaluated by RT-qPCR. Moreover, the method is easily scalable and implemented in conventional laboratories as a low cost and suitable alternative to automated systems for the detection of SARS-CoV-2. The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly reached pandemic levels. Sufficient testing for SARS-CoV-2 has remained essential for tracking and containing the virus. SARS-CoV-2 testing capabilities are still limited in many countries. Here, we explore the use of conventional RNA purification as an alternative to automated systems for detection of SARS-CoV-2 by RT-qPCR. 87 clinical swab specimens were extracted by conventional phenol-chloroform RNA purification and compared to commercial platforms for RNA extraction and the fully integrated Cobas®6800 diagnostic system. Our results show that the conventional RNA extraction is fully comparable to modern automated systems regarding analytical sensitivity and specificity with respect to detection of SARS-CoV-2 as evaluated by RT-qPCR. Moreover, the method is easily scalable and implemented in conventional laboratories as a low cost and suitable alternative to automated systems for the detection of SARS-CoV-2.

The diagnosis of mycobacterial infections has been dramatically improved by the introduction of molecular methods aimed to reduce the time to diagnosis as compared with culture. The broad range pan-mycobacterial PCR can detect all the mycobacterial species directly from clinical specimens. We aimed to evaluate its usefulness and its clinical added value for the diagnosis of nontuberculous mycobacterial (NTM) infections. We performed a retrospective study (2003–2013) including 952 samples taken from 639 patients with clinical suspicion of NTM infection. The performance of smear microscopy, PCR and culture was established using clinical data to investigate discrepant results. We also compared the time to microbial diagnosis between the direct PCR and culture. The sensitivity, specificity, positive and negative predictive values of the PCR were 61.6% (53.5–69.1), 99.1% (98.2–99.6), 92.8% (85.8–96.5) and 93.4% (91.6–94.9), respectively, when considering all specimens. When considering smear-positive specimens and smear-negative specimens, the sensitivity was 81.6% and 40%, respectively. The sensitivity for pulmonary and extra-pulmonary smear-positive specimens was 85.2% versus 72.7%. The median time to identification at species level was 35 days (SD, 17.67) for culture and 6 days (SD, 2.67) for the PCR (when positive), which represents a 29-day shorter time to results (p

Objectives Screening for methicillin-resistant Staphylococcus aureus (MRSA) is part of many recommendations to control MRSA. Several rapid PCR tests are available commercially and updated versions are constantly released. We aimed to evaluate the performance of three consecutive versions (G3, Gen3 and NxG) of the XpertMRSA test. Methods Routine samples for MRSA screening were simultaneously tested by culture and rapid PCR. The three versions of XpertMRSA were used successively and compared with culture. Results A total of 3512, 2794 and 3288 samples were analysed by culture and by the G3, Gen3 and NxG XpertMRSA versions, respectively. The rates of positive-by-culture in the three groups were 5.0%, 4.7% and 4.3%, respectively. The sensitivity improved over time (71.4, 95% CI 64.0–77.9; 82.3, 95% CI 74.4–88.2; and 84.3%, 95% CI 77.0–89.7, respectively), but not significantly. The specificity (98.4, 95% CI 97.9–98.8; 96.8, 95% CI 96.0–97.4; and 99.1, 95% CI 98.7–99.4, respectively) and the positive likelihood ratios (45.7, 95% CI 34.4–60.8; 25.6, 95% CI 20.5–32.0; and 97.1, 95% CI 66.3–142.4) were significantly lower in the Gen3 version (p Conclusions These significant differences in performance show the importance of evaluating each new version of a commercial test.

Pneumonia is a severe infectious disease. In addition to common viruses and bacterial pathogens (e.g. Streptococcus pneumoniae), fastidious respiratory pathogens like Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella spp. can cause severe atypical pneumonia. They do not respond to penicillin derivatives, which may cause failure of antibiotic empirical therapy. The same applies for infections with B. pertussis and B. parapertussis, the cause of pertussis disease, that may present atypically and need to be treated with macrolides. Moreover, these fastidious bacteria are difficult to identify by culture or serology, and therefore often remain undetected. Thus, rapid and accurate identification of bacterial pathogens causing atypical pneumonia is crucial. We performed a retrospective method evaluation study to evaluate the diagnostic performance of the new, commercially available Lightmix ® multiplex RT-PCR assay that detects these fastidious bacterial pathogens causing atypical pneumonia. In this retrospective study, 368 clinical respiratory specimens, obtained from patients suffering from atypical pneumonia that have been tested negative for the presence of common agents of pneumonia by culture and viral PCR, were investigated. These clinical specimens have been previously characterized by singleplex RT-PCR assays in our diagnostic laboratory and were used to evaluate the diagnostic performance of the respiratory multiplex Lightmix ® RT-PCR. The multiplex RT-PCR displayed a limit of detection between 5 and 10 DNA copies for different in-panel organisms and showed identical performance characteristics with respect to specificity and sensitivity as in-house singleplex RT-PCRs for pathogen detection. The Lightmix ® multiplex RT-PCR assay represents a low-cost, time-saving and accurate diagnostic tool with high throughput potential. The time-to-result using an automated DNA extraction device for respiratory specimens followed by multiplex RT-PCR detection was below 4 h, which is expected to significantly improve diagnostics for atypical pneumonia-associated bacterial pathogens.

Funding: This study was funded by the World Bank under the East Africa Public Health Laboratory Networking Project (EAPHLNP). Background: Xpert MTB/RIF assay is a highly sensitive test for TB diagnosis, but still costly to most low-income countries. Several implementation strategies instead of frontline have been suggested; however with scarce data. We assessed accuracy of different Xpert MTB/RIF implementation strategies to inform national roll-out. Methods: This was a cross-sectional study of 1,924 adult presumptive TB patients in five regional referral hospitals of Uganda. Two sputum samples were collected, one for fluorescent microscopy (FM) and Xpert MTB/RIF examined at the study site laboratories. The second sample was sent to the Uganda Supra National TB reference laboratory for culture using both Lowenstein Jensen (LJ) and liquid culture (MGIT). We compared the sensitivities of FM, Xpert MTB/RIF and the incremental sensitivity of Xpert MTB/RIF among patients negative on FM using LJ and/or MGIT as a reference standard. Results: A total 1924 patients were enrolled of which 1596 (83%) patients had at least one laboratory result and 1083 respondents had a complete set of all the laboratory results. A total of 328 (30%) were TB positive on LJ and /or MGIT culture. The sensitivity of FM was n (%; 95% confidence interval) 246 (63.5%; 57.9-68.7) overall compared to 52 (55.4%; 44.1-66.3) among HIV positive individuals, while the sensitivity of Xpert MTB/RIF was 300 (76.2%; 71.7-80.7) and 69 (71.6%; 60.5-81.1) overall and among HIV positive individuals respectively. Overall incremental sensitivity of Xpert MTB/RIF was 60 (36.5%; 27.7-46.0) and 20 (41.7%; 25.5-59.2) among HIV positive individuals. Conclusion: Xpert MTB/RIF has a higher sensitivity than FM both in general population and HIV positive population. Xpert MTB/RIF offers a significant increase in terms of diagnostic sensitivity even when it is deployed selectively i.e. among smear negative presumptive TB patients. Our results support frontline use of Xpert MTB/RIF assay in high HIV/TB prevalent countries. In settings with limited access, mechanisms to refer smear negative sputum samples to Xpert MTB/RIF hubs are recommended. Publisher PDF

Objective: To determine the current use and potential acceptance (by tuberculosis experts worldwide) of novel rapid tests for the diagnosis of tuberculosis that are in line with World Health Organization target product profiles. Methods: A multilingual survey was disseminated online between July and November of 2016. Results: A total of 723 individuals from 114 countries responded to the survey. Smear microscopy was the most commonly used rapid tuberculosis test (available to 90.9% of the respondents), followed by molecular assays (available to 70.7%). Only a small proportion of the respondents in middle- and low-income countries had access to interferon-gamma-release assays. Serological and lateral flow immunoassays were used by more than a quarter (25.4%) of the respondents. Among the respondents who had access to molecular tests, 46.7% were using the Xpert assay overall, that proportion being higher in lower middle-income countries (55.6%) and low-income countries (76.6%). The data also suggest that there was some alignment of pricing for molecular assays. Respondents stated they would accept novel rapid tuberculosis tests if available, including molecular assays (acceptable to 86.0%) or biomarker-based serological assays (acceptable to 81.7%). Simple biomarker-based assays were more commonly deemed acceptable in middle- and low-income countries. Conclusions: Second-generation molecular assays have become more widely available in high- and low-resource settings. However, the development of novel rapid tuberculosis tests continues to be considered important by tuberculosis experts. Our data also underscore the need for additional training and education of end users. RESUMO Objetivo: Determinar o uso atual e a aceitação potencial (por especialistas em tuberculose em todo o mundo) de novos testes rápidos para o diagnóstico de tuberculose que estão alinhados com os perfis de produtos alvo da Organização Mundial da Saúde. Métodos: Um inquérito multilingue foi divulgado on-line entre julho e novembro de 2016. Resultados: Um total de 723 indivíduos de 114 países respondeu ao inquérito. A baciloscopia foi o teste rápido para tuberculose mais utilizado (disponível para 90,9% dos entrevistados), seguida de ensaios moleculares (disponível para 70,7%). Apenas uma pequena proporção dos entrevistados de países de renda média e baixa tinha acesso a ensaios de liberação de IFN-γ. Imunoensaios de fluxo lateral e testes sorológicos eram utilizados por mais de um quarto dos entrevistados (25,4%). Entre os entrevistados que tinham acesso a testes moleculares, 46,7% utilizavam o teste Xpert de forma geral, sendo essa proporção maior em países de renda média baixa (55,6%) e renda baixa (76,6%). Os dados também sugerem que houve algum alinhamento de preços para testes moleculares. Os entrevistados afirmaram que aceitariam novos testes rápidos para tuberculose, se disponíveis, incluindo testes moleculares (aceitáveis para 86,0%) ou testes sorológicos baseados em biomarcadores (aceitáveis para 81,7%). Testes simples baseados em biomarcadores foram mais comumente considerados aceitáveis nos países de renda média e baixa. Conclusões: Os testes moleculares de segunda geração tornaram-se mais amplamente disponíveis em locais tanto com poucos quanto com muitos recursos. No entanto, o desenvolvimento de novos testes rápidos para tuberculose continua a ser considerado importante por especialistas em tuberculose. Nossos dados também ressaltam a necessidade de maior formação e educação dos usuários finais.

Urine samples are increasingly used for diagnosing infections including Escherichia coli, Ebola virus, and Zika virus. However, extraction and concentration of nucleic acid biomarkers from urine is necessary for many molecular detection strategies such as polymerase chain reaction (PCR). Since urine samples typically have large volumes with dilute biomarker concentrations making them prone to false negatives, another impediment for urine-based diagnostics is the establishment of appropriate controls particularly to rule out false negatives. In this study, a mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) DNA target was added to retrospectively collected urine samples from tuberculosis (TB)-infected and TB-uninfected patients to indicate extraction of intact DNA and removal of PCR inhibitors from urine samples. We tested this design on surrogate urine samples, retrospective 1 milliliter (mL) urine samples from patients in Lima, Peru and retrospective 5 mL urine samples from patients in Cape Town, South Africa. Extraction/PCR control DNA was detectable in 97% of clinical samples with no statistically significant differences among groups. Despite the inclusion of this control, there was no difference in the amount of TB IS6110 Tr-DNA detected between TB-infected and TB-uninfected groups except for samples from known HIV-infected patients. We found a increase in TB IS6110 Tr-DNA between TB/HIV co-infected patients compared to TB-uninfected/HIV-infected patients (N=18, p=0.037). The inclusion of an extraction/PCR control DNA to indicate successful DNA extraction and removal of PCR inhibitors should be easily adaptable as a sample preparation control for other acellular sample types.

Background: The Xpert (TM) MTB/RIF (XP) has a higher sensitivity than sputum smear microscopy (70% versus 35%) for TB diagnosis and has been endorsed by the WHO for TB high burden countries to increase case finding among HIV co-infected presumptive TB patients. Its impact on the diagnosis of smear-negative TB in a routine care setting is unclear. We determined the change in diagnosis, treatment and mortality of smear-negative presumptive TB with routine use of Xpert MTB/RIF (XP). Methods: Prospective cohort study of HIV-positive smear-negative presumptive TB patients during a 12-month period after XP implementation in a well-staffed and trained integrated TB/HIV clinic in Kampala, Uganda. Prior to testing clinicians were asked to decide whether they would treat empirically prior to Xpert result; actual treatment was decided upon receipt of the XP result. We compared empirical and XP-informed treatment decisions and all-cause mortality in the first year. Results: Of 411 smear-negative presumptive TB patients, 175 (43%) received an XP; their baseline characteristics did not differ. XP positivity was similar in patients with a pre-XP empirical diagnosis and those without (9/29 [17%] versus 14/142 [10%], P = 0.23). Despite XP testing high levels of empirical treatment prevailed (18%), although XP results did change who ultimately was treated for TB. When adjusted for CD4 count, empirical treatment was not associated with higher mortality compared to no or microbiologically confirmed treatment. Conclusions: XP usage was lower than expected. The lower sensitivity of XP in smear-negative HIV-positive patients led experienced clinicians to use XP as a "rule-in" rather than "rule-out" test, with the majority of patients still treated empirically.

The advances in molecular biology of the last decades have dramatically improved the field of diagnostic bacteriology. In particular, PCR-based technologies have impacted the diagnosis of infections caused by obligate intracellular bacteria such as pathogens from the Chlamydiacae family. Here, we describe a real-time PCR-based method using the Taqman technology for the diagnosis of Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia abortus infection. The method presented here can be applied to various clinical samples and can be adapted on opened molecular diagnostic platforms.

Objective: To determine the current use and potential acceptance (by tuberculosis experts worldwide) of novel rapid tests for the diagnosis of tuberculosis that are in line with World Health Organization target product profiles. Methods: A multilingual survey was disseminated online between July and November of 2016. Results: A total of 723 individuals from 114 countries responded to the survey. Smear microscopy was the most commonly used rapid tuberculosis test (available to 90.9% of the respondents), followed by molecular assays (available to 70.7%). Only a small proportion of the respondents in middle- and low-income countries had access to interferon-gamma-release assays. Serological and lateral flow immunoassays were used by more than a quarter (25.4%) of the respondents. Among the respondents who had access to molecular tests, 46.7% were using the Xpert assay overall, that proportion being higher in lower middle-income countries (55.6%) and low-income countries (76.6%). The data also suggest that there was some alignment of pricing for molecular assays. Respondents stated they would accept novel rapid tuberculosis tests if available, including molecular assays (acceptable to 86.0%) or biomarker-based serological assays (acceptable to 81.7%). Simple biomarker-based assays were more commonly deemed acceptable in middle- and low-income countries. Conclusions: Second-generation molecular assays have become more widely available in high- and low-resource settings. However, the development of novel rapid tuberculosis tests continues to be considered important by tuberculosis experts. Our data also underscore the need for additional training and education of end users.

Outbreaks of Streptococcus pyogenes hypervirulent clones are constant public health threats. In western Switzerland, an increase of severe cases of S. pyogenes invasive infections was observed between December 2015 and March 2016. Our aim was (i) to investigate these cases by the use of Whole Genome Sequencing (WGS) and (ii) to determine the specific virulome and resistome of each isolate in order to undertake adequate public health measures. Eleven Streptococcus pyogenes strains isolated from 11 patients with severe invasive infections between December 13, 2015 and March 12, 2016 were included in our study. Practically, emm-typing, MLST and WGS were used to investigate the relatedness between the isolates. The presence of virulence and antibiotic resistance genes as well as mutations in transcriptional regulators of virulence and in genes encoding for antibiotic targets were assessed. Three and two groups of isolates shared the same emm-type and ST type, respectively. Single Nucleotide Polymorphism (SNP) analysis revealed 14 to 32 SNPs between the strains of the same emm-type group, ruling out the possibility of a clonal outbreak. Mutations found in covS and rocA could partially explain an increased virulence. As these reassuring results were obtained in less than 10 days, no specific hospital hygiene and no dedicated public health measures had to be undertaken. WGS is a powerful technique to discriminate between closely related strains, excluding an outbreak in less than 10 days. Moreover, WGS provided extensive data on the virulome and resistome of all these strains. Electronic supplementary material The online version of this article (doi:10.1007/s10096-017-2905-z) contains supplementary material, which is available to authorized users.

Objectives Blood culture results inadequately stratify the mortality risk in critically ill patients with sepsis. We sought to establish the prognostic significance of the presence of microbial DNA in the bloodstream of patients hospitalized with suspected sepsis. Methods We analysed the data collected during the Rapid Diagnosis of Infections in the Critically Ill (RADICAL) study, which compared a novel culture-independent PCR/electrospray ionization-mass spectrometry (ESI-MS) assay with standard microbiological testing. Patients were eligible for the study if they had suspected sepsis and were either hospitalized or were referred to one of nine intensive care units from six European countries. The blood specimen for PCR/ESI-MS assay was taken along with initial blood culture taken for clinical indications. Results Of the 616 patients recruited to the RADICAL study, 439 patients had data on outcome, results of the blood culture and PCR/ESI-MS assay available for analysis. Positive blood culture and PCR/ESI-MSI result was found in 13% (56/439) and 40% (177/439) of patients, respectively. Either a positive blood culture (p 0.01) or a positive PCR/ESI-MS (p 0.005) was associated with higher SOFA scores on enrolment to the study. There was no difference in 28-day mortality observed in patients who had either positive or negative blood cultures (35% versus 32%, p 0.74). However, in patients with a positive PCR/ESI-MS assay, mortality was significantly higher in comparison to those with a negative result (42% versus 26%, p 0.001). Conclusions Presence of microbial DNA in patients with suspected sepsis might define a patient group at higher risk of death.

Airborne precautions are required at hospital admission for patients with suspected pulmonary tuberculosis. The isolation is maintained until 3 serially collected sputum smears are acid-fast bacilli negative, a time- and labor-intensive method with limited sensitivity and specificity, which has a great impact on patient flow management. We evaluated the possibility of replacing the result of microscopy by the semiquantitative result of the molecular point-of-care test Xpert MTB/RIF to assess patients' transmission risk to quickly guide airborne isolation decisions in low-endemic countries. The performance of the Xpert MTB/RIF, used as a first-line test, was compared to the results of microscopy for specimens (n=242) collected from May 2010 to December 2014 in Lausanne, Switzerland. The sensitivity and specificity of Xpert MTB/RIF were 91.5% (65/71) and 99.6% (170/171), respectively, vs. 64.8% (46/71) and 94.2% (161/171) for microscopy. Samples with negative Xpert MTB/RIF were all smear negative for Mycobacterium tuberculosis (negative predictive value, 100%). The semiquantitative results of Xpert MTB/RIF—high, medium, low or very low—were found to correlate with acid-fast bacilli detection: positive predictive value of 100% (6/6), 96.5% (27/28), 52.2% (12/23) and 11.1% (1/9) respectively. Finally, when including clinical criteria, we identified 11 smear-negative but Xpert MTB/RIF–positive patients with a significant transmission potential. In conclusion, our data support the introduction of an Xpert MTB/RIF–based strategy as a replacement of smear microscopy for a faster and more accurate management of tuberculosis patients' transmission risk in a low-prevalence country.

Currently available nucleic acid amplification platforms for tuberculosis (TB) detection are not designed to be simple or inexpensive enough to implement in decentralized settings in countries with a high burden of disease. The loop-mediated isothermal amplification platform (LAMP) may change this. We conducted a study in adults with symptoms suggestive of TB in India, Uganda, and Peru to establish the feasibility of using TB-LAMP (Eiken Chemical Co.) in microscopy laboratories compared with using smear microscopy against a reference standard of solid and liquid cultures. Operational characteristics were evaluated as well. A total of 1,777 participants met the eligibility criteria and were included for analysis. Overall, TB-LAMP sensitivities among culture-positive samples were 97.2% (243/250; 95% confidence interval [CI], 94.3% to 98.2%) and 62.0% (88/142; 95% CI, 53.5% to 70.0%) for smear-positive and smear-negative TB, respectively, but varied widely by country and operator. Specificities ranged from 94.5% (446/472; 95% CI, 92.0% to 96.4%) to 98.0% (350/357; 95% CI, 96.0% to 99.2%) by country. A root cause analysis identified high temperatures, high humidity, and/or low reaction volumes as possible causes for false-positive results, as they may result in nonspecific amplification. The study was repeated in India with training focused on vulnerable steps and an updated protocol; 580 participants were included for analysis. Specificity in the repeat trial was 96.6% (515/533; 95% CI, 94.7% to 97.9%). To achieve acceptable performance of LAMP at the microscopy center level, significant training and infrastructure requirements are necessary.

Cutaneous and mucosal leishmaniasis is widely distributed in Central and South America. Leishmania of the Viannia subgenus are the most frequent species infecting humans. L. (V.) braziliensis, L. (V.) panamensis are also responsible for metastatic mucosal leishmaniasis. Conventional or real time PCR is a more sensitive diagnostic test than microscopy, but the cost and requirement for infrastructure and trained personnel makes it impractical in most endemic regions. Primary health systems need a sensitive and specific point of care (POC) diagnostic tool. We developed a novel POC molecular diagnostic test for cutaneous leishmaniasis caused by Leishmania (Viannia) spp. Parasite DNA was amplified using isothermal Recombinase Polymerase Amplification (RPA) with primers and probes that targeted the kinetoplast DNA. The amplification product was detected by naked eye with a lateral flow (LF) immunochromatographic strip. The RPA-LF had an analytical sensitivity equivalent to 0.1 parasites per reaction. The test amplified the principal L. Viannia species from multiple countries: L. (V.) braziliensis (n = 33), L. (V.) guyanensis (n = 17), L. (V.) panamensis (n = 9). The less common L. (V.) lainsoni, L. (V.) shawi, and L. (V.) naiffi were also amplified. No amplification was observed in parasites of the L. (Leishmania) subgenus. In a small number of clinical samples (n = 13) we found 100% agreement between PCR and RPA-LF. The high analytical sensitivity and clinical validation indicate the test could improve the efficiency of diagnosis, especially in chronic lesions with submicroscopic parasite burdens. Field implementation of the RPA-LF test could contribute to management and control of cutaneous and mucosal leishmaniasis., Author Summary Cutaneous leishmaniasis is a parasitic disease transmitted by the bite of sandflies that produces skin ulcers. The severe, disfiguring form of the disease is characterized by parasite dissemination to the mucosa of the nose and palate. Current diagnosis is based on microscopy which has low sensitivity in chronic cases. Molecular methods (PCR) that detect parasite DNA are highly sensitive but costs and personnel training make it impossible to implement it in resource-limited areas. We developed a novel molecular method (RPA-LF) that could be applied in the field because it does not require sophisticated equipment. It is also very sensitive and specific to detect the principal Leishmania species that produce cutaneous leishmaniasis in Latin America. Future field implementation of RPA-LF could have a positive impact on disease management and control.

Acute fever is one of the most common presenting symptoms globally. In order to reduce the empiric use of antimicrobial drugs and improve outcomes, it is essential to improve diagnostic capabilities. In the absence of microbiology facilities in low-income settings, an assay to distinguish bacterial from non-bacterial causes would be a critical first step. To ensure that patient and market needs are met, the requirements of such a test should be specified in a target product profile (TPP). To identify minimal/optimal characteristics for a bacterial vs. non-bacterial fever test, experts from academia and international organizations with expertise in infectious diseases, diagnostic test development, laboratory medicine, global health, and health economics were convened. Proposed TPPs were reviewed by this working group, and consensus characteristics were defined. The working group defined non-severely ill, non-malaria infected children as the target population for the desired assay. To provide access to the most patients, the test should be deployable to community health centers and informal health settings, and staff should require 90% and >80% for sensitivity and specificity, respectively. Other key characteristics, to account for the challenging environment at which the test is targeted, included: i) time-to-result

Molecular diagnosis using real-time polymerase chain reaction (PCR) may allow earlier diagnosis of rickettsiosis. We developed a duplex real-time PCR that amplifies (1) DNA of any rickettsial species and (2) DNA of both typhus group rickettsia, that is, Rickettsia prowazekii and Rickettsia typhi. Primers and probes were selected to amplify a segment of the 16S rRNA gene of Rickettsia spp. for the pan-rickettsial PCR and the citrate synthase gene (gltA) for the typhus group rickettsia PCR. Analytical sensitivity was 10 copies of control plasmid DNA per reaction. No cross-amplification was observed when testing human DNA and 22 pathogens or skin commensals. Real-time PCR was applied to 16 clinical samples. Rickettsial DNA was detected in the skin biopsies of three patients. In one patient with severe murine typhus, the typhus group PCR was positive in a skin biopsy from a petechial lesion and seroconversion was later documented. The two other patients with negative typhus group PCR suffered from Mediterranean and African spotted fever, respectively; in both cases, skin biopsy was performed on the eschar. Our duplex real-time PCR showed a good analytical sensitivity and specificity, allowing early diagnosis of rickettsiosis among three patients, and recognition of typhus in one of them.

Background: There is an urgent need to identify molecular signatures in small cell lung cancer (SCLC) that may select patients who are likely to respond to molecularly targeted therapies. In this study, we investigate the feasibility of undertaking focused molecular analyses on routine diagnostic biopsies in patients with SCLC.Methods: A series of histopathologically confirmed formalin-fixed, paraffin-embedded SCLC specimens were analysed for epidermal growth factor receptors (EGFR), KRAS, NRAS and BRAF mutations, ALK gene rearrangements and MET amplification. EGFR and KRAS mutation testing was evaluated using real time polymerase chain reaction (RT-PCR cobas®), BRAF and NRAS mutations using multiplex PCR and capillary electrophoresis-single strand conformation analysis, and ALK and MET aberrations with fluorescent in situ hybridization. All genetic aberrations detected were validated independently.Results: A total of 105 patients diagnosed with SCLC between July 1990 and September 2006 were included. 60 (57 %) patients had suitable tumour tissue for molecular testing. 25 patients were successfully evaluated for all six pre-defined molecular aberrations. Eleven patients failed all molecular analysis. No mutations in EGFR, KRAS and NRAS were detected, and no ALK gene rearrangements or MET gene amplifications were identified. A V600E substitution in BRAF was detected in a Caucasian male smoker diagnosed with SCLC with squamoid and glandular features.Conclusion: The paucity of patients with sufficient tumour tissue, quality of DNA extracted and low frequency of aberrations detected indicate that alternative molecular characterisation approaches are necessary, such as the use of circulating plasma DNA in patients with SCLC.

The nose is the anatomical site usually recommended for methicillin-resistant Staphylococcus aureus (MRSA) screening. Other sites are also recommended, but are more controversial. We showed that the sensitivities of MRSA detection from nasal swabs alone were 48% and 62% by culture or by rapid PCR test, respectively. These percentages increased to 79% and 92% with the addition of groin swabs, and to 96% and 99% with the addition of groin and throat swabs. In conclusion, neither by culture nor by rapid PCR test is nose sampling alone sufficient for MRSA detection. Additional anatomical sites should include at least the groin and throat.

High-throughput sequencing (HTS) provides the means to analyze clinical specimens in unprecedented molecular detail. While this technology has been successfully applied to virus discovery and other related areas of research, HTS methodology has yet to be exploited for use in a clinical setting for routine diagnostics. Here, a bioinformatics pipeline (ezVIR) was designed to process HTS data from any of the standard platforms and to evaluate the entire spectrum of known human viruses at once, providing results that are easy to interpret and customizable. The pipeline works by identifying the most likely viruses present in the specimen given the sequencing data. Additionally, ezVIR can generate optional reports for strain typing, can create genome coverage histograms, and can perform cross-contamination analysis for specimens prepared in series. In this pilot study, the pipeline was challenged using HTS data from 20 clinical specimens representative of those most often collected and analyzed in daily practice. The specimens (5 cerebrospinal fluid, 7 bronchoalveolar lavage fluid, 5 plasma, 2 serum, and 1 nasopharyngeal aspirate) were originally found to be positive for a diverse range of DNA or RNA viruses by routine molecular diagnostics. The ezVIR pipeline correctly identified 14 of 14 specimens containing viruses with genomes of

Due to overuse, we are about to reach the end of the antibiotic era. Each of us is responsible to limit their usage to a minimum. Respiratory infections are the first cause of antibiotic prescriptions. The use of new simple tests available at the bedside can be very useful in this context. The development of sophisticated molecular diagnostic tools, such as "multiorganism" panels, may revolutionize our approach to respiratory infections. The key will be to interpret the results correctly, with due consideration of the statement, "Treat patients, not lab results".

The performance of the Xpert MRSA polymerase chain reaction (PCR) assay on pooled nose, groin, and throat swabs (three nylon flocked eSwabs into one tube) was compared to culture by analyzing 5,546 samples. The sen- sitivity (0.78, 95 % confidence interval (CI) 0.73-0.82) and specificity (0.99, 95 % CI 0.98-0.99) were similar to the results from published studies on separated nose or other specimens. Thus, the performance of the Xpert MRSA assay was not affected by pooling the three specimens into one assay, allowing a higher detection rate without increasing laboratory costs, as compared to nose samples alone.

To systematically review and estimate pooled sensitivity and specificity of the polymerase chain reaction (PCR) technique compared to recommended reference tests in the diagnosis of suspected syphilis at various stages and in various biological materials.

Conventional tube culture systems have long been the mainstay in clinical virology for the growth and identification of viruses from clinical specimens. Innovations such as centrifugation-enhanced shell vial and multiwell plate cultures and the use of genetically engineered and mixed cell lines, coupled with faster detection of viral replication, have allowed for reasonable turnaround times for even some of the most slowly growing clinically important human viruses. However, molecular methods, in particular, the PCR, have usurped the role of viral culture in many laboratories, limiting the use of this traditional method of virus detection or replacing it altogether. Advances and improvements in molecular technology over time have also resulted in newer generations of more rapid and accurate molecular assays for the detection, quantification, and genetic characterization of viruses. For this point-counterpoint, we have asked two individuals, Richard L. Hodinka of the Children's Hospital of Philadelphia, a clinical virologist whose laboratory has completely eliminated viral culture in favor of molecular methods, and Laurent Kaiser, head of the Virology Laboratory at the University of Geneva Hospital, who continues to be a strong advocate of viral culture, to discuss the relevance of viral culture in the molecular age.

In 2010, Jaton et al . (False-negative PCR result due to gene polymorphism: the example of Neisseria meningitidis . J Clin Microbiol 2010;48:4590–2) reported an isolate of Neisseria meningitidis serogroup B that was not detected by the ctrA quantitative real-time PCR (qRT-PCR) used in our diagnostic laboratory. Sequence analysis of ctrA revealed several single nucleotide polymorphisms responsible for the negative qRT-PCR. Therefore, we sequenced the genome of this isolate and performed comparative genomics to propose new gene targets for the specific detection of N. meningitidis from clinical specimens. We identified 11 genes as specific to N. meningitidis genomes and common to at least 177 (97%) of the 183 genomes available. Among them, three genes ( metA, tauE and shlA ) were selected to develop new qRT-PCRs for the detection of N. meningitidis DNA. The three qRT-PCRs were highly sensitive and specific, and they exhibited a good reproducibility when tested on plasmidic positive controls and genomic DNA extracted from strains of N. meningitidis and other relevant bacterial species. The clinical sensitivity and specificity of metA and tauE qRT-PCRs were both 100% based on a testing of cerebrospinal fluid samples positive for N. meningitidis or other clinically relevant bacteria. Despite a 100% specificity, the sensitivity of the shlA qRT-PCR was only 70%. We thus recommend using the metA and/or tauE qRT-PCRs developed here. To prevent PCR failure in the presence of new polymorphic strains, the detection of dual targets by duplex qRT-PCR would be more accurate and suitable for the diagnosis of N. meningitidis from clinical specimens.

Rickettsia and other intracellular bacteria: recent outbreaks, novel pathogens, emerging diseases, new tools, and outstanding discoveries

Group B streptococcus (GBS) is a leading cause of infectious neonatal morbidity and mortality. Timely and accurate identification of colonized mothers is imperative so that antibioprophylaxis can be implemented during labour to reduce the risk of neonatal sepsis. We planned our study to analyse the diagnostic accuracy of an intrapartum PCR assay to identify GBS-colonized women and to allow the implementation of correct (i.e. at least 4 h) intrapartum antibiotic prophylaxis based on the PCR results. We included 695 women in labour who were tested for rectovaginal GBS carriage by culture and PCR. Women were also screened at 35–37 weeks of gestation. Intrapartum GBS colonization was 19.3%. Assay sensitivity was 81.0% for antenatal culture and 85.0% for intrapartum PCR; p 0.72. GBS colonization (n = 107) was known at least 4 h before delivery in 68 (64%) and 73 (68%) women based on antenatal culture and intrapartum PCR, respectively. Among 43 women delivering preterm, correct status was known at least 4 h before delivery in 10 (23%) and 32 (74%) women according to antenatal culture and intrapartum PCR, respectively. These results support the concept that GBS screening can be performed routinely during labour in a clinical setting. The intrapartum approach is at least as accurate as the antenatal screening, with the additional advantage of identifying women delivering preterm or not followed during pregnancy.

Initial evaluations of the Cobas AmpliPrep/Cobas TaqMan human immunodeficiency virus type 1 (HIV-1) test (CAP/CTM) demonstrated good performance but, afterwards, reports about underquantification were published. We investigated whether the problem was solved with a second version of this assay, the Cobas AmpliPrep/Cobas TaqMan HIV-1 test, version 2.0 (CAP/CTM v2.0). The remaining plasma of 375 consecutive HIV-1 positive samples with a viral load of ≥4,000 copies/ml was collected in three laboratories. The samples were diluted and retested with our routine method Cobas AmpliPrep/Cobas Amplicor HIV-1 monitor test v1.5 in ultrasensitive mode (CAP/CA PHS), as well as with the CAP/CTM and CAP/CTM v2.0 tests. An absolute difference between the results of two methods of ≥0.71 log10copies/ml was defined as moderately discrepant, and an absolute difference of ≥0.93 log10copies/ml was defined as severely discrepant. In addition, criteria for considering the new methods equivalent to the routine method were formulated. (i) For CAP/CTM compared to CAP/CA PHS, 36 (9.5%) and 20 (5.3%) samples were, respectively, considered moderately and severely underquantified by CAP/CTM. The mean difference between CAP/CTM and CAP/CA PHS was −0.32 log10copies/ml. Eight of nineteen of the severely underquantified samples were from patients infected with HIV-1 subtype B strain. (ii) For CAP/CTM v2.0 compared to CAP/CA PHS, no sample was moderately or severely underquantified by CAP/CTM v2.0. A mean difference of 0.08 log10copies/ml was found with CAP/CTM v2.0 compared to CAP/CA PHS. The underquantification problem of the CAP/CTM kit was clearly demonstrated. The criteria for the equivalence of CAP/CTM v2.0 to the routine test CAP/CA PHS were fulfilled.

International audience; Hydrometrocolpos and polydactyly diagnosed in the prenatal period or early childhood may raise diagnostic dilemmas especially in distinguishing McKusick-Kaufman syndrome (MKKS) and the Bardet-Biedl syndrome (BBS). These two conditions can initially overlap. With time, the additional features of BBS appearing in childhood, such as retinitis pigmentosa, obesity, learning disabilities and progressive renal dysfunction allow clear differentiation between BBS and MKKS. Genotype overlap also exists, as mutations in the MKKS-BBS6 gene are found in both syndromes. We report 7 patients diagnosed in the neonatal period with hydrometrocolpos and polydactyly who carry mutations in various BBS genes (BBS6, BBS2, BBS10, BBS8 and BBS12), stressing the importance of wide BBS genotyping in patients with this clinical association for diagnosis, prognosis and genetic counselling.

PURPOSE: To assess the prognostic and predictive significance of p53 and Bcl-2 protein expression in high risk patients with breast cancer treated with dose-dense sequential chemotherapy. PATIENTS AND METHODS: From June 1997 until November 2000, 595 patients were randomized to three cycles of epirubicin (E) 110 mg/m2 followed by three cycles of paclitaxel (P) 250 mg/m2 followed by three cycles of 'intensified' CMF (cyclophosphamide 840 mg/m2, methotrexate 47 mg/m2 and fluorouracil 840 mg/m2) or to four cycles of E, followed by four cycles of CMF. p53 and Bcl-2 expression was investigated by immunohistochemistry in 392 and 397 patients respectively. RESULTS: Positive expression of p53 was detected in 104 (26.5%) patients and was significantly associated with negative hormonal status, worse histologic grade, higher incidence of disease relapse and higher rate of death. p53 positive expression was a significant negative predictor of overall survival (OS) (P = 0.002) and disease-free survival (DFS) (P = 0.001). Negative expression of Bcl-2 was detected in 203 (51%) patients and was significantly associated with negative hormonal status. Multivariate analysis revealed that, positive p53 expression, higher number of positive nodes and worse tumor grade were related to significantly poorer OS and DFS. CONCLUSIONS: For both treatments, p53 positive expression was a significant negative prognostic factor for OS and DFS while Bcl-2 was not. No predictive ability of p53 status or Bcl-2 status for paclitaxel treatment was evident. Ann Oncol

During the 6th International Meeting on Rickettsia and Rickettsial Diseases that was held in Heraklion (Greece) from 5–7 June, 2011, a large number of important recent discoveries were presented, some of which are available in this Thematic Issue as original articles. These included significant contributions to the understanding of the pathogenesis, epidemiology, diagnosis, and clinical presentation of rickettsial diseases as well as human and animal infections by various other intracellular bacteria, such as Anaplasma , Bartonella , Coxiella , Ehrlichia , Waddlia , and Wolbachia . Most of these bacteria are strict intracellular bacteria that are particularly difficult to study, owing to their dependence on the host cell and to the lack of straightforward systems to manipulate their genomes. Rather …

The accurate microbiological diagnosis of diarrhoea involves numerous laboratory tests and, often, the pathogen is not identified in time to guide clinical management. With next-generation sequencing (NGS) becoming cheaper, it has huge potential in routine diagnostics. The aim of this study was to evaluate the potential of NGS-based diagnostics through direct sequencing of faecal samples. Fifty-eight clinical faecal samples were obtained from patients with diarrhoea as part of the routine diagnostics at Hvidovre University Hospital, Denmark. Ten samples from healthy individuals were also included. DNA was extracted from faecal samples and sequenced on the Illumina MiSeq system. Species distribution was determined with MGmapper and NGS-based diagnostic prediction was performed based on the relative abundance of pathogenic bacteria and Giardia and detection of pathogen-specific virulence genes. NGS-based diagnostic results were compared to conventional findings for 55 of the diarrhoeal samples; 38 conventionally positive for bacterial pathogens, two positive for Giardia, four positive for virus and 11 conventionally negative. The NGS-based approach enabled detection of the same bacterial pathogens as the classical approach in 34 of the 38 conventionally positive bacterial samples and predicted the responsible pathogens in five of the 11 conventionally negative samples. Overall, the NGS-based approach enabled pathogen detection comparable to conventional diagnostics and the approach has potential to be extended for the detection of all pathogens. At present, however, this approach is too expensive and time-consuming for routine diagnostics.

BACKGROUND: The Xpert™ MTB/RIF (XP) has a higher sensitivity than sputum smear microscopy (70% versus 35%) for TB diagnosis and has been endorsed by the WHO for TB high burden countries to increase case finding among HIV co-infected presumptive TB patients. Its impact on the diagnosis of smear-negative TB in a routine care setting is unclear. We determined the change in diagnosis, treatment and mortality of smear-negative presumptive TB with routine use of Xpert MTB/RIF (XP). METHODS: Prospective cohort study of HIV-positive smear-negative presumptive TB patients during a 12-month period after XP implementation in a well-staffed and trained integrated TB/HIV clinic in Kampala, Uganda. Prior to testing clinicians were asked to decide whether they would treat empirically prior to Xpert result; actual treatment was decided upon receipt of the XP result. We compared empirical and XP-informed treatment decisions and all-cause mortality in the first year. RESULTS: Of 411 smear-negative presumptive TB patients, 175 (43%) received an XP; their baseline characteristics did not differ. XP positivity was similar in patients with a pre-XP empirical diagnosis and those without (9/29 [17%] versus 14/142 [10%], P = 0.23). Despite XP testing high levels of empirical treatment prevailed (18%), although XP results did change who ultimately was treated for TB. When adjusted for CD4 count, empirical treatment was not associated with higher mortality compared to no or microbiologically confirmed treatment. CONCLUSIONS: XP usage was lower than expected. The lower sensitivity of XP in smear-negative HIV-positive patients led experienced clinicians to use XP as a "rule-in" rather than "rule-out" test, with the majority of patients still treated empirically.

Occurrence of non-Candida albicans Candida (NCAC) species that are associated with elevated MIC values and therapeutic failures are increasing. As a result, timely and accurate means of identification to the species level is becoming an essential part of diagnostic practices in clinical settings. In this study, 301 clinically isolated yeast strains recovered from various anatomical sites [Blood (n = 145), other sites (n = 156)] were used to assess the accuracy and practicality of API 20C AUX and 21-plex PCR compared to MALDI-TOF MS and large subunit rDNA (LSU rDNA). MALDI-TOF MS correctly identified 98.33% of yeast isolates, 100% of top five Candida species, 95.7% of rare yeast species, while 1.3% of isolates were misidentified. API 20C AUX correctly identified 83.7% of yeast isolates, 97.2% of top five Candida species, 61.8% of rare yeast species, while 16.2% of yeast isolates were misidentified. The 21-plex PCR, accurately identified 87.3% of yeast isolates, 100% of top five Candida species, 72% of rare yeast species, but it misidentified 1.3% of rare yeast species while 9.9% of whole yeast isolates were not identified. The combination of rapidity of 21-plex PCR and comprehensiveness of API 20C AUX, led to correct identification of 92% of included yeast isolates. Due to expensiveness of MALDI-TOF MS and sequencing, this combination strategy could be the most accurate and inexpensive alternative identification strategy for developing countries. Moreover, by the advent and development of cost-effective, reliable, and rapid PCR machines that cost 130 US dollars, 21-plex could be integrated in routine laboratories of developing and resource-limited countries to specifically identify 95% causative agents of yeast-related infections in human. Databases of MALDI-TOF MS, API 20C AUX, and the number of target species identified by 21-plex require further improvement to keep up with the diverse spectrum of yeast species.