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Schirmer strips and conjunctival swabs are used in ophthalmology for the collection of tears and fluids. One of the biggest challenges during the COVID-19 pandemic has been accurate diagnosis and, in some cases, ocular manifestations are among the first symptoms. In this context, this study aimed to collect evidence to support the use of Schirmer strips and conjunctival swabs as a method of sample collection for viral analysis. A literature search was conducted following the Scoping Review protocol defined by The Joanna Briggs Institute. Studies were analyzed regarding virus research, collection methods, and sample analysis. The findings support that viruses can be detected on the ocular surface through analysis of Schirmer strips and conjunctival swabs. However, additional studies with larger samples and time data are necessary to confirm these conclusions. RESUMO A fita de Schirmer e o swab conjunctival são utilizados na oftalmologia como métodos de coleta para lágrimas e fluidos. Durante a pandemia da COVID-19, um dos desafios foi o diagnóstico correto e se sabe que, em alguns casos, as manifestações oculares podem ser um dos primeiros sintomas. Nesse contexto, este estudo tem como objetivo levantar evidência que destaque o uso de fitas de Schirmer e de swabs conjuntivais como método de coleta para análise viral. Conduziu-se uma revisão de literatura seguindo o protocolo para Scoping Review definido pelo Joanna Briggs Institute. Os pesquisadores analisaram os estudos em busca do vírus pesquisado, os métodos de coleta e os métodos de análise. Vírus podem ser detectados na superfície ocular através da análise de fitas de Schirmer e de swabs conjuntivais, entretanto novos estudos com populações maiores e com definições claras de tempo são necessários para conclusões mais assertivas no tema.

Testing for disease-related DNA methylation changes provides clinically relevant information in personalized patient care. Methylation-Sensitive High-Resolution Melting (MS-HRM) is a method used for measuring methylation changes and has already been used in diagnostic settings. This method utilizes one set of primers that initiate the amplification of both methylated and non-methylated templates. Therefore, the quantification of the methylation levels using MS-HRM is hampered by the PCR bias phenomenon. Some approaches have been proposed to calculate the methylation level of samples using the high-resolution melting (HRM) curves. However, limitations of the methylation calculation using MS-HRM have not been evaluated systematically and comprehensively. We used the Area Under the Curve (AUC), a derivative of the HRM curves, and least square approximation (LSA) to establish a procedure that allowed us to infer methylation levels in an MS-HRM experiment and assess the limitations of that procedure for the assays’ specific methylation level measurement. The developed procedure allowed, with certain limitations, estimation of the methylation levels using HRM curves.

Objective: To explore factors associated with a high vaginal GBS load during labor considering (1) the recto-vaginal GBS load at 35–37 weeks’ gestation determined by culture and (2) the vaginal GBS colonization determined by a polymerase chain reaction (PCR) assay during labor. Methods: From an unselected cohort of 902 pregnant women, we obtained (1) recto-vaginal swabs for culture of GBS at 35–37 weeks’ gestation (GBSrectovag-36), (2) vaginal swabs for GBS PCR detection at labor (PCRvag-labor), and (3) vaginal swabs for culture of GBS at labor (GBSvag-labor). The GBS load was classified semi quantitatively according to a culture protocol without prior broth enrichment of the swab samples: none (0), few (+), some (++), or many (+++) GBS colonies. Results: Among 902 unselected pregnant women, 859 (95%) had a vaginal swab culture taken at labor, which was classified semi quantitatively. High load GBSvag-labor (+++) were found in 31 participants. GBSrectovag-36 showed a sensitivity of 90% (28/31) and a PPV of 23% (28/121), whereas PCRvag-labor had a sensitivity of 98% (30/31, non-significant difference) and a PPV of 42% (30/71, p

Schirmer strips and conjunctival swabs are used in ophthalmology for the collection of tears and fluids. One of the biggest challenges during the COVID-19 pandemic has been accurate diagnosis and, in some cases, ocular manifestations are among the first symptoms. In this context, this study aimed to collect evidence to support the use of Schirmer strips and conjunctival swabs as a method of sample collection for viral analysis. A literature search was conducted following the Scoping Review protocol defined by The Joanna Briggs Institute. Studies were analyzed regarding virus research, collection methods, and sample analysis. The findings support that viruses can be detected on the ocular surface through analysis of Schirmer strips and conjunctival swabs. However, additional studies with larger samples and time data are necessary to confirm these conclusions.

Use of telomere length (TL) as a biomarker for various environmental exposures and diseases has increased in recent years. Various methods have been developed to measure telomere length. Polymerase chain reaction (PCR)-based methods remain wide-spread for population-based studies due to the high-throughput capability. While several studies have evaluated the repeatability and reproducibility of different TL measurement methods, the results have been variable. We conducted a literature review of TL measurement cross-method comparison studies that included a PCR-based method published between January 1, 2002 and May 25, 2020. A total of 25 articles were found that matched the inclusion criteria. Papers were reviewed for quality of methodologic reporting of sample and DNA quality, PCR assay characteristics, sample blinding, and analytic approaches to determine final TL. Overall, methodologic reporting was low as assessed by two different reporting guidelines for qPCR-based TL measurement. There was a wide range in the reported correlation between methods (as assessed by Pearson’s r) and few studies utilized the recommended intra-class correlation coefficient (ICC) for assessment of assay repeatability and methodologic comparisons. The sample size for nearly all studies was less than 100, raising concerns about statistical power. Overall, this review found that the current literature on the relation between TL measurement methods is lacking in validity and scientific rigor. In light of these findings, we present reporting guidelines for PCR-based TL measurement methods and results of analyses of the effect of assay repeatability (ICC) on statistical power of cross-sectional and longitudinal studies. Additional cross-laboratory studies with rigorous methodologic and statistical reporting, adequate sample size, and blinding are essential to accurately determine assay repeatability and replicability as well as the relation between TL measurement methods.

The Covid-19 pandemic imposes new diagnostic strategies in order to optimize the medical care of our patients. The current biblio-graphy, although of low quality, shows a sensitivity of 56 to 83 % for the Covid-19 PCR. Even though one negative test can exclude a Covid-19 in the majority of cases, the NPV (Negative Predictive Value) decreases with increasing prevalence (pre-test probability). This finding suggests the need for strict auto-isolation of patients until the resolution of their symptoms. For patients that present with typical symptoms, who have a presumed Covid-19 prevalence of -40-50 %, a negative test should be interpreted with caution and a repeat test may be needed.

Background Genitourinary tuberculosis is the third most common form of extrapulmonary tuberculosis. Diagnosis is difficult because of unspecific clinical manifestations and low accuracy of conventional tests. Unfortunately, the delayed diagnosis impacts the urinary tract severely. Nucleic acid amplification tests yield fast results, and among these, new technologies can also detect drug resistance. There is lack of consensus regarding the use of these tests in genitourinary tuberculosis; we therefore aimed to assess the accuracy of nucleic acid amplification tests in the diagnosis of genitourinary tuberculosis and to evaluate the heterogeneity between studies. Methods We did a systematic review and meta-analysis of research articles comparing the accuracy of a reference standard and a nucleic acid amplification test for diagnosis of urinary tract tuberculosis. We searched Medline, EMBASE, Web of Science, LILACS, Cochrane Library, and Scopus for articles published between Jan 1, 1990, and Apr 14, 2016. Two investigators identified eligible articles and extracted data for individual study sites. We analyzed data in groups with the same index test. Then, we generated pooled summary estimates (95% CIs) for sensitivity and specificity by use of random-effects meta-analysis when studies were not heterogeneous. Results We identified eleven relevant studies from ten articles, giving information on PCR, LCR and Xpert MTB/RIF tests. All PCR studies were “in-house” tests, with different gene targets and had several quality concerns therefore we did not proceed with a pooled analysis. Only one study used LCR. Xpert studies were of good quality and not heterogeneous, pooled sensitivity was 0·87 (0·66–0·96) and specificity was 0·91 (0·84–0·95). Conclusion PCR studies were highly heterogeneous. Among Xpert MTB/RIF studies, specificity was favorable with an acceptable confidence interval, however new studies can update meta-analysis and get more precise estimates. Further high-quality studies are urgently needed to improve diagnosis of genitourinary tuberculosis. Protocol registration PROSPERO CRD42016039020. Electronic supplementary material The online version of this article (doi:10.1186/s12879-017-2476-8) contains supplementary material, which is available to authorized users.

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

Observational studies are a basis for much of our knowledge of veterinary pathology, yet considerations for conducting pathology-based observational studies are not readily available. In part 1 of this series, we offered advice on planning and carrying out an observational study. Part 2 of the series focuses on methodology. Our general recommendations are to consider using already-validated methods, published guidelines, data from primary sources, and quantitative analyses. We discuss 3 common methods in pathology research—histopathologic scoring, immunohistochemistry, and polymerase chain reaction—to illustrate principles of method validation. Some aspects of quality control include use of clear objective grading criteria, validation of key reagents, assessing sample quality, determining specificity and sensitivity, use of technical and biologic negative and positive controls, blinding of investigators, approaches to minimizing operator-dependent variation, measuring technical variation, and consistency in analysis of the different study groups. We close by discussing approaches to increasing the rigor of observational studies by corroborating results with complementary methods, using sufficiently large numbers of study subjects, consideration of the data in light of similar published studies, replicating the results in a second study population, and critical analysis of the study findings.

The global emergence of azole-resistant Aspergillus fumigatus strains is a growing public health concern. Different patterns of azole resistance are linked to mutations in cyp51A Therefore, accurate characterization of the mechanisms underlying azole resistance is critical to guide selection of the most appropriate antifungal agent for patients with aspergillosis. This study describes a new sequencing-free molecular screening tool for early detection of the most frequent mutations known to be associated with azole resistance in A. fumigatus PCRs targeting cyp51A mutations at positions G54, Y121, G448, and M220 and targeting different tandem repeats (TRs) in the promoter region were designed. All PCRs were performed simultaneously, using the same cycling conditions. Amplicons were then distinguished using a high-resolution melting assay. For standardization, 30 well-characterized azole-resistant A. fumigatus strains were used, yielding melting curve clusters for different resistance mechanisms for each target and allowing detection of the most frequent azole resistance mutations, i.e., G54E, G54V, G54R, G54W, Y121F, M220V, M220I, M220T, M220K, and G448S, and the tandem repeats TR34, TR46, and TR53 Validation of the method was performed using a blind panel of 80 A. fumigatus azole-susceptible or azole-resistant strains. All strains included in the blind panel were properly classified as susceptible or resistant with the developed method. The implementation of this screening method can reduce the time needed for the detection of azole-resistant A. fumigatus isolates and therefore facilitate selection of the best antifungal therapy in patients with aspergillosis. This study was financed by a research project from the Fondo de Investigación Sanitaria (FIS) (project PI13/02145). It was supported by Plan Nacional de I+D+i and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, and the Spanish Network for Research in Infectious Diseases (REIPI grant RD12/0015), cofinanced by the European Development Regional Fund. L.B.-M. has a research contract from the Spanish Network for Research in Infectious Diseases (REIPI grant RD12/0015). O.R.-M. holds a predoctoral fellowship from the Fondo de Investigaciones Sanitarias (grant FI14CIII/00025). S.G. was supported by a research fellowship from the Fondo de Investigaciones Sanitarias of the Spanish Ministry of Science and Innovation (grant FI10/00464). E.M. was supported by a project from the Spanish Fondo de Investigación Sanitaria (FIS grant PI15CIII/00019). Sí

Expanded CAG/CTG repeats underlie the aetiology of 14 neurological and neuromuscular disorders. The size of the repeat tract determines in large part the severity of these disorders with longer tracts causing more severe phenotypes. Expanded CAG/CTG repeats are also unstable in somatic tissues, which is thought to modify disease progression. Routine molecular biology applications involving these repeats, including quantifying their instability, are plagued by low PCR yields. This leads to the need for setting up more PCRs of the same locus, thereby increasing the risk of carry-over contamination. Here we aimed to reduce this risk by pre-treating the samples with a Uracil N-Glycosylase (Ung) and using dUTP instead of dTTP in PCRs. We successfully applied this method to the PCR amplification of expanded CAG/CTG repeats, their sequencing, and their molecular cloning. In addition, we optimized the gold-standard method for measuring repeat instability, small-pool PCR (SP-PCR), such that it can be used together with Ung and dUTP-containing PCRs, without compromising data quality. We performed SP-PCR on myotonic-dystrophy-derived samples containing an expansion as large as 1000 repeats, demonstrating the applicability to clinically-relevant material. Thus, we expect the protocols herein to be applicable for molecular diagnostics of expanded repeat disorders.

Massive parallel sequencing (MPS) can accurately quantify mitochondrial DNA (mtDNA) single nucleotide variants (SNVs), but no MPS methods are currently validated to simultaneously and accurately establish the breakpoints and frequency of large deletions at low heteroplasmic loads. Here we present the thorough validation of an MPS protocol to quantify the load of very low frequency, large mtDNA deletions in bulk DNA and single cells, along with SNV calling by standard methods. We used a set of well-characterized DNA samples, DNA mixes and single cells to thoroughly control the study. We developed a custom script for the detection of mtDNA rearrangements that proved to be more accurate in detecting and quantifying deletions than pre-existing tools. We also show that PCR conditions and primersets must be carefully chosen to avoid biases in the retrieved variants and an increase in background noise, and established a lower detection limit of 0.5% heteroplasmic load for large deletions, and 1.5 and 2% for SNVs, for bulk DNA and single cells, respectively. Finally, the analysis of different single cells provided novel insights into mtDNA cellular mosaicism.European Journal of Human Genetics advance online publication, 23 August 2017; doi:10.1038/ejhg.2017.129.

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.

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.

In regenerating liver, hepatocytes accumulate lipids before the major wave of parenchymal growth. This transient, regeneration-associated steatosis (TRAS) is required for liver recovery, but its purpose is unclear. The tumor suppressor phosphatase and tensin homolog (PTEN) is a key inhibitor of the protein kinase B/mammalian target of rapamycin axis that regulates growth and metabolic adaptations after hepatectomy. In quiescent liver, PTEN causes pathological steatosis when lost, whereas its role in regenerating liver remains unknown. Here, we show that PTEN down-regulation promotes liver growth in a TRAS-dependent way. In wild-type mice, PTEN reduction occurred after TRAS formation, persisted during its disappearance, and correlated with up-regulated β-oxidation at the expense of lipogenesis. Pharmacological modulation revealed an association of PTEN with TRAS turnover and hypertrophic liver growth. In liver-specific Pten(-/-) mice shortly after induction of knockout, hypertrophic regeneration was accelerated and led to hepatomegaly. The resulting surplus liver mass was functional, as demonstrated by raised survival in a lethal model of resection-induced liver failure. Indirect calorimetry revealed lipid oxidation as the primary energy source early after hepatectomy. The shift from glucose to lipid usage was pronounced in Pten(-/-) mice and correlated with the disappearance of TRAS. Partial inhibition of β-oxidation led to persisting TRAS in Pten(-/-) mice and abrogated hypertrophic liver growth. PTEN down-regulation may promote β-oxidation through β-catenin, whereas hypertrophy was dependent on mammalian target of rapamycin complex 1. CONCLUSION PTEN down-regulation after hepatectomy promotes the burning of TRAS-derived lipids to fuel hypertrophic liver regeneration. Therefore, the anabolic function of PTEN deficiency in resting liver is transformed into catabolic activities upon tissue loss. These findings portray PTEN as a node coordinating liver growth with its energy demands and emphasize the need of lipids for regeneration. (Hepatology 2017;66:908-921).

OBJECTIVE: The purpose of this study was to investigate if oropharyngeal swab polymerase chain reaction (PCR) could predict osteoarticular infection (OAI) due to Kingella kingae in young children. METHODS: One hundred twenty-three consecutive children aged 6 to 48 months presenting with atraumatic osteoarticular complaints were prospectively studied. All had a clinical evaluation, imaging, and blood samples. Blood and oropharyngeal specimens were tested with a PCR assay specific for K kingae. OAI was defined as bone, joint, or blood detection of pathogenic bacteria, or MRI consistent with infection in the absence of positive microbiology. K kingae OAI was defined by blood, bone, or synovial fluid positivity for the organism by culture or PCR. RESULTS: Forty children met the OAI case definition; 30 had K kingae OAI, 1 had another organism, and 9 had no microbiologic diagnosis. All 30 oropharyngeal swabs from the K kingae case patients and 8 swabs from the 84 patients without OAI or with OAI caused by another organism were positive. The sensitivity and specificity of the oropharyngeal swab PCR assay for K kingae were 100% and 90.5%, respectively. CONCLUSIONS: Detection of K kingae DNA in oropharyngeal swabs of children with clinical findings of OAI is predictive of K kingae OAI. If these findings are replicated in other settings, detection of K kingae by oropharyngeal swab PCR could improve the recognition of OAI.

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.

The H275Y and E119D neuraminidase (NA) mutations constitute important molecular markers of resistance to NA inhibitors in A(H1N1) pdm09 viruses. We used reverse transcriptase-droplet digital PCR amplification (RT-ddPCR) to analyze quasi-species at codons 275 and 119 of the NA in A(H1N1) pdm09 viruses recovered from an immuncompromised patient who received oseltamivir and zanamivir therapies. RT-ddPCR assays detected and quantified H275Y and E119D mutations with an efficiency that was comparable to that of high throughput sequencing (HiSeq 2500 Illumina, San Diego, CA) technology. With its sensitivity and reproducibility, RT-ddPCR could be a reliable method for accurate detection and quantification of major NAI-resistance mutations in clinical settings. J. Med. Virol. 89:737-741, 2017. © 2016 Wiley Periodicals, Inc.

BACKGROUND: Foot infections are a major cause of morbidity in people with diabetes and the most common cause of diabetes-related hospitalization and lower extremity amputation. Staphylococcus aureus is by far the most frequent species isolated from these infections. In particular, methicillin-resistant S. aureus (MRSA) has emerged as a major clinical and epidemiological problem in hospitals. MRSA strains have the ability to be resistant to most β-lactam antibiotics, but also to a wide range of other antimicrobials, making infections difficult to manage and very costly to treat. To date, there are two fifth-generation cephalosporins generally efficacious against MRSA, ceftaroline and ceftobripole, sharing a similar spectrum. Biofilm formation is one of the most important virulence traits of S. aureus. Biofilm growth plays an important role during infection by providing defence against several antagonistic mechanisms. In this study, we analysed the antimicrobial susceptibility patterns of biofilm-producing S. aureus strains isolated from diabetic foot infections. The antibiotic minimum inhibitory concentration (MIC) was determined for ten antimicrobial compounds, along with the minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC), followed by PCR identification of genetic determinants of biofilm production and antimicrobial resistance. RESULTS: Results demonstrate that very high concentrations of the most used antibiotics in treating diabetic foot infections (DFI) are required to inhibit S. aureus biofilms in vitro, which may explain why monotherapy with these agents frequently fails to eradicate biofilm infections. In fact, biofilms were resistant to antibiotics at concentrations 10-1000 times greater than the ones required to kill free-living or planktonic cells. The only antibiotics able to inhibit biofilm eradication on 50 % of isolates were ceftaroline and gentamicin. CONCLUSIONS: The results suggest that the antibiotic susceptibility patterns cannot be applied to biofilm established infections. Selection of antimicrobial therapy is a critical step in DFI and should aim at overcoming biofilm disease in order to optimize the outcomes of this complex pathology.

Peritonsillar abscess (PTA) is the most frequent complication of acute tonsillitis and a prevalent cause for acute admission to otorhinolaryngology departments. Our aim was to examine the role of viruses in the pathogenesis of PTA, as this has not previously been considered. We examined both palatine tonsils from 25 patients undergoing acute tonsillectomy for PTA, using PCR-based assays for herpes simplex virus-1 and -2 (HSV-1 and -2), adenovirus, Epstein-Barr virus (EBV), influenza A and B, and respiratory syncytial virus (RSV) A and B. We similarly examined tonsils from 55 patients undergoing elective tonsillectomy due to chronic tonsillar conditions. These patients served as a control group, as they did not have a clinically apparent infection at the time of surgery. Only HSV-1 (5/80, 6.3%), adenovirus (11/80, 13.8%), and EBV (71/80, 88.8%) were detected in our study population. There was no statistically significant difference in the frequency of these viruses across different diagnostic groups. Quantification of EBV load demonstrated no differences between the PTA and the elective tonsillectomy group, nor between the abscessed and non-abscessed tonsil of PTA patients. In summary, our data do not support a significant role for the examined viruses in the pathogenesis of PTA. Peritonsillar abscess (PTA) is the most frequent complication of acute tonsillitis and a prevalent cause for acute admission to otorhinolaryngology departments. Our aim was to examine the role of viruses in the pathogenesis of PTA, as this has not previously been considered. We examined both palatine tonsils from 25 patients undergoing acute tonsillectomy for PTA, using PCR-based assays for herpes simplex virus-1 and -2 (HSV-1 and -2), adenovirus, Epstein-Barr virus (EBV), influenza A and B, and respiratory syncytial virus (RSV) A and B. We similarly examined tonsils from 55 patients undergoing elective tonsillectomy due to chronic tonsillar conditions. These patients served as a control group, as they did not have a clinically apparent infection at the time of surgery. Only HSV-1 (5/80, 6.3%), adenovirus (11/80, 13.8%), and EBV (71/80, 88.8%) were detected in our study population. There was no statistically significant difference in the frequency of these viruses across different diagnostic groups. Quantification of EBV load demonstrated no differences between the PTA and the elective tonsillectomy group, nor between the abscessed and non-abscessed tonsil of PTA patients. In summary, our data do not support a significant role for the examined viruses in the pathogenesis of PTA.

Purpose The aim of this article is to report a case of uveal tuberculoma simulating an intraocular tumor in which the diagnosis was only possible by polymerase chain reaction (PCR) analysis. Method This is a case report. Results A36-year-old male presented with a progressive growing intraocular tumor and no history or positive test for other systemic disease. The eye eventually turned blind and painful and was enucleated. Histopathologic analysis revealed a granulomatous reaction and caseation necrosis but failed to identify any causative microorganisms. Final tuberculosis diagnosis was only possible by quantitative PCR. Conclusion Isolated uveal tuberculoma can present in an otherwise healthy patient with negative systemic tuberculosis evaluation. PCR can be used to confirm tuberculosis when other methods have failed.

Background BK virus infections can have clinically significant consequences in immunocompromised individuals. Detection and monitoring of active BK virus infections in certain situations is recommended and therefore PCR assays for detection of BK virus have been developed. The performance of current BK PCR detection assays is limited by the existence of viral polymorphisms, unknown at the time of assay development, resulting in inconsistent detection of BK virus. The objective of this study was to identify a stable region of the BK viral genome for detection by PCR that would be minimally affected by polymorphisms as more sequence data for BK virus becomes available. Results Employing a combination of techniques, including amino acid and DNA sequence alignment and interspecies analysis, a conserved, stable PCR target region of the BK viral genomic region was identified within the VP2 gene. A real-time quantitative PCR assay was then developed that is specific for BK virus, has an analytical sensitivity of 15 copies/reaction (450 copies/ml) and is highly reproducible (CV ≤ 5.0%). Conclusion Identifying stable PCR target regions when limited DNA sequence data is available may be possible by combining multiple analysis techniques to elucidate potential functional constraints on genomic regions. Applying this approach to the development of a real-time quantitative PCR assay for BK virus resulted in an accurate method with potential clinical applications and advantages over existing BK assays.

Ninety-one patients were studied serially for chimeric status following allogeneic stem cell transplantation (SCT) for severe aplastic anaemia (SAA) or Fanconi Anaemia (FA). Short tandem repeat polymerase chain reaction (STR-PCR) was used to stratify patients into five groups: (A) complete donor chimeras (n = 39), (B) transient mixed chimeras (n = 15) (C) stable mixed chimeras (n = 18), (D) progressive mixed chimeras (n = 14) (E) recipient chimeras with early graft rejection (n = 5). As serial sampling was not possible in Group E, serial chimerism results for 86 patients were available for analysis. The following factors were analysed for association with chimeric status: age, sex match, donor type, aetiology of aplasia, source of stem cells, number of cells engrafted, conditioning regimen, graft-versus-host disease (GvHD) prophylaxis, occurrence of acute and chronic GvHD and survival. Progressive mixed chimeras (PMCs) were at high risk of late graft rejection (n = 10, P < 0.0001). Seven of these patients lost their graft during withdrawal of immunosuppressive therapy. STR-PCR indicated an inverse correlation between detection of recipient cells post-SCT and occurrence of acute GvHD (P = 0.008). PMC was a bad prognostic indicator of survival (P = 0.003). Monitoring of chimeric status during cyclosporin withdrawal may facilitate therapeutic intervention to prevent late graft rejection in patients transplanted for SAA.

BACKGROUND: Various studies have suggested the involvement of infectious agents in chronic inflammatory diseases, including atherosclerosis. By using a novel subtraction broad-range PCR approach, we defined bacterial DNA signatures in surgically removed sterile abdominal aorta samples of patients with aortic atherosclerosis.METHODS: Partial bacterial 16S rDNA nucleotide sequences were determined using broad-range PCR from aortic samples of 20 patients, and from appropriate methodological controls. In all, 160 sequences from 16 clone libraries were studied.RESULTS: After subtraction analysis 16 clinically relevant bacterial sequence-types were identified among the patient samples, whereas 29 were discarded as potential methodological contaminants. On average 2.2+/-1.2 different bacterial sequence-types were present in the nine true PCR-positive atheroma samples.CONCLUSIONS: Many studies have reported the presence of a variety of bacterial sequences from atherosclerotic lesions. However, the results obtained with these PCR technologies may have been skewed by methodological contaminants. After our subtraction approach, 63% of the remaining sequence-types from sites of aortic atherosclerosis were related to those of known human pathogens. This may imply that advanced atherosclerotic plaques accumulate bacterial DNA.

An accurate amplified fragment length polymorphism (AFLP) method, including three primer sets for the selective amplification step, was developed to display the phylogenetic position of Photobacterium isolates collected from salmon products. This method was efficient for discriminating the three species Photobacterium phosphoreum, Photobacterium iliopiscarium and Photobacterium kishitanii, until now indistinctly gathered in the Photobacterium phosphoreum species group known to be strongly responsible for seafood spoilage. The AFLP fingerprints enabled the isolates to be separated into two main clusters that, according to the type strains, were assigned to the two species P. phosphoreum and P. iliopiscarium. P. kishitanii was not found in the collection. The accuracy of the method was validated by using gyrB-gene sequencing and luxA-gene PCR amplification, which confirmed the species delineation. Most of the isolates of each species were clonally distinct and even those that were isolated from the same source showed some diversity. Moreover, this AFLP method may be an excellent tool for genotyping isolates in bacterial communities and for clarifying our knowledge of the role of the different members of the Photobacterium species group in seafood spoilage.

Drug susceptibility testing using molecular techniques can enhance the identification of drug-resistant Mycobacterium tuberculosis. Two multiplex real-time polymerase chain reaction (qPCR) assays were developed to detect the most common resistance-associated mutations to isoniazid (katGS315T, inhA-15C -> T), and rifampicin (rpoBH526Y and rpoBS531L). To assess the species specificity of the qPCR, we selected 31 nontuberculous mycobacteria (NTM) reference strains belonging to 17 species from the public collection of mycobacterial cultures (BCCM/ITM). Additionally, we tested 17 isoniazid and/or rifampicin-resistant strains with other mutations in the target genes to assess mutation specificity. The limit of detection for all the targeted mutations was 20 bacilli/reaction. Multiplex 1 showed 90%, 95%, and 100% efficiency for wild type (WT), Mut katGS315T, and Mut rpoBS531L, respectively; whereas Multiplex 2 showed 97%, 94%, and 90% efficiency for WT, Mut inhA-15, and Mut rpoBH526Y, respectively. Three of 17 strains that presented other mutations in the target genes were identified as rifampicin resistant and only 3/31 NTM showed a similar melting temperature to rpoBL531 and/or katG11.315 mutants. Thus, our proposed cascade of specific tuberculosis detection followed by drug resistance testing showed sensitivities for katGS315T, rpoBS531L, rpoBH526Y, and inhA-15 detection of 100%, 100%, 100%, and 96%, respectively; and specificities of 98%, 95%, 100%, and 100, respectively.

Monitoring of BCR-ABL transcripts has become established practice in the management of chronic myeloid leukemia. However, nucleic acid amplification techniques are prone to variations which limit the reliability of real-time quantitative PCR (RQ-PCR) for clinical decision making, highlighting the need for standardization of assays and reporting of minimal residual disease (MRD) data. We evaluated a lyophilized preparation of a leukemic cell line (K562) as a potential quality control reagent. This was found to be relatively stable, yielding comparable respective levels of ABL, GUS and BCR-ABL transcripts as determined by RQ-PCR before and after accelerated degradation experiments as well as following 5 years storage at -20 degrees C. Vials of freeze-dried cells were sent at ambient temperature to 22 laboratories on four continents, with RQ-PCR analyses detecting BCR-ABL transcripts at levels comparable to those observed in primary patient samples. Our results suggest that freeze-dried cells can be used as quality control reagents with a range of analytical instrumentations and could enable the development of urgently needed international standards simulating clinically relevant levels of MRD.

Background Methicillin-resistance in staphylococci is conferred by the mecA gene, located on the genomic island Staphylococcal Cassette Chromosome mec (SCCmec). SCCmec mobility relies on the Ccr recombinases, which catalyze insertion and excision form the host’s chromosome. Although being a crucial step in its horizontal transfer, little is known about the dynamics of SCCmec excision. Results A quantitative PCR-based method was used to measure the rate of SCCmec excision by amplifying the chromosome–chromosome junction and the circularized SCCmec resulting from excision. SCCmec excision rate was measured in methicillin-resistant Staphylococcus aureus (MRSA) strain N315 at various growth times in broth cultures. In the present experimental settings, excision of SCCmec occurred at a rate of approximately 2 × 10−6 in MRSA N315. Conclusion This work brings new insights in the poorly understood SCCmec excision process. The results presented herein suggest a model in which excision occurs during a limited period of time at the early stages of growth. Electronic supplementary material The online version of this article (doi:10.1186/s13104-015-1815-3) contains supplementary material, which is available to authorized users.

The availability of rapid and reliable infectious disease diagnostics that can provide results directly from patient specimens represents a major unmet need in managing critically ill patients. Current sepsis guidelines recommend initiation of IV antibiotic therapy as early as possible, ideally within the first hour (1), as any delay in effective antimicrobial therapy may result in decreased survival (2). Effective therapy requires that the identity of causative pathogens and their resistance patterns are known. However, the current standard-of-care, which depends on blood culture-based initial diagnosis, often takes at least 48–72 hours to provide a result. Furthermore, cultures often remain negative even when bacterial or fungal infections are strongly suspected (3), in part, related to concurrent antibiotic treatment (4). Molecular diagnostic techniques that do not depend on growth of organisms in culture may offer a distinct advantage over current methods. Most of the recently described molecular methods, however, rely on culture amplification as a precursor to diagnosis (5–8). Although these techniques may accelerate diagnosis for positive cultures, they do not address the significant proportion of false-negative cultures observed in patients with sepsis. In addition, many of these methods also use targeted pathogen detection with limited pathogen coverage such that negative results are often not highly predictive. Polymerase chain reaction followed by electrospray ionization-mass spectrometry (PCR/ESI-MS) can detect more than 800 bloodstream infection (BSI)-relevant pathogens in a single assay and in approximately 6 hours (9–13). It can also identify three classes of antibiotic resistance markers associated with resistance to methicillin (mecA), vancomycin (vanA/vanB), and carbapenems (KPC). Using this technique, we recently demonstrated 83% sensitivity and 94% specificity compared with culture for direct detection of pathogens in whole-blood specimens from patients with suspected BSIs (13). Here, we describe findings from the multicenter observational Rapid Diagnosis of Infections in the Critically Ill (RADICAL) study. The primary objective was to compare results obtained using the novel culture-independent PCR/ESI-MS technology with those obtained from standard microbiological testing as a measure of clinical performance. Secondarily, to broadly address the clinical value of PCR/ESI-MS detections, a panel of independent clinical adjudicators was used to identify changes in patient management that may have occurred had the results from the PCR/ESI-MS technology been available for clinical use and assumed to be correct.

Fasciola hepatica is a zoonotic infection with a worldwide distribution. Autochthonous cases have not been reported in the Amazon region of Peru. Operculated eggs resembling F. hepatica were identified in the stools of five out of 215 subjects in a remote indigenous community of the Peruvian jungle. Polymerase chain reaction targeting Fasciola hepatica cytochrome oxidase subunit 1 (COI) gene and sequencing of the products confirmed Fasciola infection.

Rapid aetiological diagnosis of bacterial meningitis is crucial for the early targeting of antimicrobial and adjuvant therapy. Broad-range polymerase chain reaction (PCR) targeting the 16S rRNA gene allows aetiological diagnosis of bacterial meningitis when applied to cerebrospinal fluid (CSF). We assessed the additional diagnostic effect of applying a novel broad-range real time PCR and subsequent DNA sequencing to culture, microscopy, and broad-range conventional PCR on CSF in patients with suspected bacterial meningitis. Broad-range conventional PCR and broad-range real time PCR with subsequent DNA sequencing were applied to 206 CSF specimens collected consecutively from 203 patients aged 6 d to 86 y. Patients' charts were reviewed for clinical information. 17 pathogens were identified by PCR and DNA sequencing or culture. Three specimens were negative by culture but positive by broad-range real time PCR. Three specimens were positive by culture but negative by broad-range real time PCR. Compared with culture, the sensitivity of broad-range real time PCR was 86%, and the specificity 98%. Conventional PCR resulted in a sensitivity of 64% and specificity of 98%. Broad-range real time PCR was generally comparable to culture of CSF and may be a useful supplement, particularly when antimicrobial therapy has been administered. Broad-range real time PCR was more sensitive than broad-range conventional PCR and microscopy.

IL-4 and IL-13 are prototypic Th2 cytokines that generate an “alternatively activated” phenotype in macrophages. We used high-density oligonucleotide microarrays to investigate the transcriptional profile induced in human monocytes by IL-13. After 8-h stimulation with IL-13, 142 genes were regulated (85 increased and 57 decreased). The majority of these genes were related to the inflammatory response and innate immunity; a group of genes related to lipid metabolism was also identified, with clear implications for atherosclerosis. In addition to characteristic markers of alternatively activated macrophages, a number of novel IL-13-regulated genes were seen. These included various pattern recognition receptors, such as CD1b/c/e, TLR1, and C-type lectin superfamily member 6. Several components of the IL-1 system were regulated. IL-1RI, IL-1RII, and IL-1Ra were all up-regulated, whereas the IL-1β-converting enzyme, caspase 1, and IRAK-M were down-regulated. LPS-inducible caspase 1 enzyme activity was also reduced in IL-13-stimulated monocytes, with a consequent decrease in pro-IL-1β processing. These data reveal that IL-13 has a potent effect on the transcriptional profile in monocytes. The IL-13-induced modulation of genes related to IL-1 clearly highlights the tightly controlled and complex levels of regulation of the production and response to this potent proinflammatory cytokine.

DNA typing was requested to investigate a presumptive cancer diagnosis error by confirming whether benign and cancerous prostatic tissue in the same presurgical haematoxylin and eosin stained slide belonged to the same person. After independent histological re-examination of the slide by a pathologist, manual slide dissection was used to guarantee independent and high recovery DNA isolation from each tissue section, avoiding carryover and background contamination. Nuclear DNA quantification performed by real time polymerase chain reaction (PCR) revealed the absence of human DNA for short tandem repeat (STR) typing. Mitochondrial DNA was only obtained by performing PCR of very short fragments ( approximately 100 bp), indicating high DNA degradation. Different low frequency hypervariable region I haplotypes were obtained from each tissue section (normal tissue section haplotype: 16224C, 16234T, 16311C, 16356C; cancer tissue section haplotype: 16256T, 16270T, 16293G). Only the normal tissue section haplotype matched that obtained from the patient's blood sample, indicating that the cancer tissue section originated from an unknown patient. These results supported the hypothesis of sample mix up during block processing or slide preparation by a carryover mechanism. Mitochondrial genetic typing is recommended to exclude the possibility of carryover artefacts when low DNA content and high degradation compromise conventional STR typing.

Dendritic arbor development of subventricular zone-derived interneurons is a critical step in their integration into functional circuits of the postnatal olfactory bulb. However, the mechanism and molecular control of this process remain unknown. In this study, we have developed a culture model where dendritic development of purified subventricular zone cells proceeds under serum-free conditions in the absence of added growth factors and non-neural cells. We demonstrate that the large majority of these cells in culture express GABA and elaborate dendritic arbors with spine-like protrusions but they do not possess axons. These neurons expressed receptors for neurotrophins including p75, TrkB and TrkC but not TrkA. Application of exogenous neurotrophins, including brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) and nerve growth factor (NGF), to cultures stimulated dendritic growth and led to more complex dendritic arbors during the initial 3 days in culture. Our results suggest that these effects are independent of Trk receptors and mediated by the p75/ceramide signaling pathway. We also show that brain-derived neurotrophic factor is the only neurotrophin that is able to influence late-phase dendritic development via TrkB receptor activation. These results suggest that dendritic arbor development of subventricular zone-derived cells may be regulated by neurotrophins through the activation of p75 and the TrkB receptor signaling pathways in a sequentially defined temporal pattern.

Cryptococcus neoformans serotype A strains commonly infect immunocompromised patients to cause fungal meningitis. To understand the basis of serotype A cryptococcal infections in apparently immunocompetent patients, we tested two hypotheses: the strains were naturally occurring hypervirulent pkr1 (PKA regulatory subunit) mutants, or the strains were hybrids with C. neoformans var. gattii strains that normally infect immunocompetent individuals. Analysis of clinical isolates obtained from apparently immunocompetent individuals from three continents revealed that none were pkr1 mutants, but several exhibited phenotypes consistent with perturbations in cAMP signaling. Additionally, none of the strains were unusual hybrids with gattii strains. Except for one strain that was an AD hybrid, all others were serotype A (var. grubii) isolates. Taken together, our findings indicate that the ability of these clinical isolates to infect apparently normal individuals may be attributable to mutations other than pkr1 and/or underlying immune system impairment in patients.

PURPOSE: Viruses of the Adenoviridae family are associated with many clinical syndromes, possessing 50 serotypes. These agents and viruses of the Herpesviridae family are the two major agents responsible for viral conjunctivitis, and a rapid diagnosis is important due to the epidemic character of adenoviral infections. METHODS: We developed a PCR without DNA extraction for adenovirus using primers that amplify a 300 bp fragment of the hexon capsid protein gene from many serotypes. RESULTS: Swab samples from cornea of seven patients with keratoconjunctivitis were analyzed, and one of them was PCR positive for adenovirus. The sequence of this fragment shows a 100% homology with the sequence of adenovirus type 8. CONCLUSION: Sequencing of 300 bp from the hexon gene allows to identify almost all Ad serotypes, including all serotypes related to epidemic keratoconjunctivitis (8,19,37) and almost all serotypes involved with Ad-associated conjunctivitis. OBJETIVO: Vírus da família Adenoviridae estão associados com muitas síndromes clínicas, sendo conhecidos 50 sorotipos. Vírus desta família e da família Herpesviridae são os maiores responsáveis por conjuntivite viral, sendo um rápido diagnóstico importante devido ao caráter epidêmico das infecções por adenovírus. MÉTODOS: Reação em cadeia da polimerase (PCR) para adenovírus foi desenvolvida utilizando iniciadores que amplificam um fragmento de 300 bp do gene da proteína hexon do capsídeo de diversos sorotipos. A reação em cadeia da polimerase foi efetuada sem a etapa de extração de DNA. RESULTADOS: Amostras de "swabs" de córneas de sete pacientes com ceratoconjuntivite foram analisadas, sendo que uma amostra foi positiva para adenovírus. O seqüenciamento deste fragmento mostrou 100% de homologia com a seqüência do adenovírus tipo 8. CONCLUSÃO: O seqüenciamento do fragmento de 300 bp do gene do hexon permite a identificação de quase todos os sorotipos de adenovírus, incluindo aqueles relacionados com a ceratoconjuntivite epidêmica (8,19,37) e todas as amostras associadas com conjuntivite.

BACKGROUND: Temporal arteritis is a primary vascular inflammatory disease. The aetiology of temporal arteritis is unknown, but the influence of environmental factors such as infections has been suggested.MATERIALS AND METHODS: We used broad-range PCR, targeting conserved regions of the gene encoding for ribosomal RNA, to detect bacterial DNA in 27 temporal artery biopsies. Five uninvolved temporal arteries were also included. A lung sample of confirmed bacterial pneumonia served as a positive control. Inflammation was examined by histochemistry and light microscopy.RESULTS: The sensitivity of the broad-range PCR assay was 5.0 fg of DNA. Bacterial DNA sequences were neither detected in 27 temporal arteritis specimens nor in the normal temporal artery samples. However, bacterial DNA was successfully amplified from the lung sample of a subject with pneumonia. In addition, human DNA was amplified by primers for human beta-actin from all clinical specimens, suggesting lack of significant inhibitors of the molecular amplification reaction. Histochemistry showed signs of strong inflammation in the arteritis samples.CONCLUSIONS: The lack of detectable amounts of bacterial DNA suggests that viable bacteria do not have a role in chronic stages of temporal arteritis. However, these findings do not rule out the possibility of bacterial degradation products as stimulants of chronic inflammation, or of viable microbes as triggering factors of acute temporal arteritis.

Early and accurate monitoring of cytomegalovirus (CMV) infection in solid-organ transplant recipients is of major importance. We have assessed the potential benefit of an ultrasensitive plasma-based PCR assay for renal transplant recipients. The pp65 CMV antigen (pp65 Ag) assay using leukocytes was employed as a routine test for the monitoring of CMV in 23 transplant recipients. We compared the pp65 antigenemia with the CMV load quantified by an ultrasensitive PCR (US-PCR) with a limit of detection of 20 CMV DNA copies/ml of plasma. CMV infection was detected in 215 (67%) of 321 plasma samples by the US-PCR compared with 124 (39%) of 321 samples by the pp65 Ag assay. The US-PCR assay permitted the detection of CMV infection episodes following transplantation a median of 12 days earlier than the pp65 Ag assay. Moreover, during CMV infection episodes, DNA detection by the US-PCR was consistently positive, whereas false negative results were frequently observed with the pp65 Ag assay. We found a good correlation between the two assays, and the peak viral loads were significantly higher in patients with CMV-related complications (median, 5,000 DNA copies/ml) than in those without symptoms (1,160 DNA copies/ml) ( P = 0.048). In addition, patients that did not require preemptive therapy based on the results of the pp65 assay had CMV loads significantly lower (median, 36 DNA copies/ml) than those that needed treatment (median, 4,703 DNA copies/ml) ( P < 0.001). These observations provided cutoff levels that could be applied in clinical practice. The ultrasensitive plasma-based PCR detected CMV infection episodes earlier and provided more consistent results than the pp65 Ag assay. This test could improve the monitoring of CMV infection or reactivation in renal transplant recipients.

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.

Treponema pallidum PCR ( Tp -PCR) testing now is recommended as a valid tool for the diagnosis of primary or secondary syphilis. The objectives were to systematically review and determine the optimal specific target gene to be used for Tp -PCR. Comparisons of the performance of the two main targets are tpp47 and polA genes were done using meta-analysis. Three electronic bibliographic databases, representing abstract books from five conferences specialized in infectious diseases from January 1990 to March 2015, were searched. Search keywords included (“syphilis” OR “ Treponema pallidum ” OR “neurosyphilis”) AND (“PCR” OR “PCR” OR “molecular amplification”). We included diagnostic studies assessing the performance of Tp -PCR targeting tpp47 ( tpp 47- Tp -PCR) or the polA gene ( polA-Tp -PCR) in ulcers from early syphilis. All studies were assessed against quality criteria using the QUADAS-2 tool. Of 37 studies identified, 62.2% were judged at low risk of bias or applicability. Most used the U.S. Centers for Disease Control and Prevention (CDC) case definitions for primary or secondary (early) syphilis (89.2%; n = 33); 15 (40.5%) used darkfield microscopy (DFM). We did not find differences in sensitivity and specificity between the two Tp -PCR methods in the subgroup of studies using adequate reference tests. Among studies using DFM as the reference test, sensitivities were 79.8% (95% confidence intervals [CI], 72.7 to 85.4%) and 71.4% (46.0 to 88.0%) for tpp47-Tp -PCR and polA-Tp -PCR ( P = 0.217), respectively; respective specificities were 95.3% (93.5 to 96.6%) and 93.7% (91.8 to 95.2%) ( P = 0.304). Our findings suggest that the two Tp -PCR methods have similar accuracy and could be used interchangeably.

Giardia duodenalis is one of the most commonly identified parasites in stool samples. Although relatively easy to treat, giardiasis can be difficult to detect as it presents similar to other diarrheal diseases. Here, we present a recombinase polymerase amplification-based Giardia (RPAG) assay to detect the presence of Giardia in stool samples. The RPAG assay was characterized on the bench top using stool samples spiked with Giardia cysts where it showed a limit-of-detection nearly as low as the gold standard polymerase chain reaction assay. The RPAG assay was then tested in the highlands of Peru on 104 stool samples collected from the surrounding communities where it showed 73% sensitivity and 95% specificity against a polymerase chain reaction and microscopy composite gold standard. Further improvements in clinical sensitivity will be needed for the RPAG assay to have clinical relevance.