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Background: Clinical diagnosis of human brucellosis (HB) is often difficult due to non-specific symptoms. Immunological tests have been the most common method used in HB diagnosis, but molecular methods based on quantitative polymerase chain reaction (qPCR) have largely replaced these diagnostic methods. The aim of this study was to validate a HB diagnostic qPCR method; assessing different target Brucella genes, and the influence of biological matrices (serum vs. whole blood) on analytical parameters., Material and Methods: Two target genes, IS711 and bcsp31, for HB molecular diagnosis were evaluated, together with biological matrix type (whole blood and serum) using samples spiked with Brucella abortus. In addition, diagnostic parameters of this qPCR method were evaluated in paired whole blood and serum samples from patients with suspected HB., Results: Both genes could be potential diagnostic targets, but IS711 showed a lower limit of detection. In spiked matrix experiments, whole blood showed a lower limit of detection than serum after probit regression (224 vs. 3681 CFU/mL) and ANOVA analysis showed a significant (p < 0.001) difference between the Cq of whole blood at all dilutions and that of serum. In 12 paired clinical samples, no serum samples and only one whole blood sample tested positive for Brucella using this qPCR detection method., Conclusions: This standardized qPCR-based Brucella detection method could improve diagnosis of HB, serving as a rapid, highly sensitive, and specific test. Whole blood is better suited to qPCR-based HB diagnosis due to the presence of higher target DNA loads in this matrix, compared to serum., (Copyright © 2021 Elsevier B.V. All rights reserved.)

It is important to develop new methods for diagnosing relapses in the co-infection of visceral leishmaniasis (VL) and HIV to enable earlier detection using less invasive methods. We report a case of a co-infected patient who had relapses after VL treatment, where the qualitative kDNA PCR showed a good performance. The kDNA PCR seems to be a useful tool for diagnosing VL and may be a good marker for predicting VL relapses after treatment of co-infected patients with clinical symptoms of the disease.

Background: Recently, there has been an increase in the incidence of cutaneous leishmaniasis (CL), which represents an important health problem. This increase may be related to the epidemiologic expansion of the infective agent and the increase in tourism in tropical areas. The difficulty in clinical diagnosis, mainly in areas in which CL is not the first consideration of local physicians, has intensified efforts to describe diagnostic tests, which should be specific, sensitive, and practical. Amongst the new tests described are those including nucleic acid amplification (polymerase chain reaction, PCR) and immunohistochemistry (IHC)., Methods: In this study, we evaluated the sensitivity of a PCR based on small subunit (SSU) ribosomal DNA, in comparison with IHC using Leishmania spp. antibodies, in biopsies embedded in paraffin., Result: The results indicated a total sensitivity of 96% (90.9% with PCR and 68.8% with IHC), showing the possibility of using paraffin-embedded biopsies to diagnose CL., Conclusion: We propose the use of the two tests together as a routine protocol for diagnosis. This would require the provision of local medical services to perform molecular biology techniques and adequate Leishmania antibodies.

Polymerase chain reaction (PCR) from bronchoalveolar lavage clinical samples (BAL) has been used to assist in the diagnosis of invasive aspergillosis. Several studies have been published regarding the utility of this test, although no systematic review of the literature has been performed to date. The objective of this systematic review was to evaluate the efficacy of PCR from BAL for the diagnosis of invasive aspergillosis in high risk patients. MEDLINE and LILACS databases (1980-2006) searches to identify articles related to PCR in diagnosis of invasive aspergillosis. For inclusion, the study had to report sufficient data to calculate sensitivity, specificity and diagnostic odds ratio of the PCR-based technique. Patients with proven and probably invasive aspergillosis were considered. Forty-five articles met our initial inclusion criteria of which 15 articles were selected. Combining the results from the different reports, the overall sensitivity and specificity values of PCR-based techniques were 79% and 94%, respectively. Contamination, specific primers and method of PCR were important variables that could complicate interpretation of these tests. The present study showed support for the clinical value of PCR from BAL for the diagnosis of invasive aspergillosis in patients with risk factors for this disease.

Background: Human brucellosis (HB) is a bacterial zoonosis that is more frequent in low income and middle-income countries; it is sometimes associated with outbreaks. The aim of this study was to describe the largest outbreak of HB in Brazil. Methods: A retrospective cohort study of patients suspected of having contracted HB in the state of Paraná, Southern Brazil from January 2009 to January 2017. Following an outbreak of 51 cases of HB in a slaughterhouse at Paiçandu in 2014, HB was defined as an obligatory reportable disease in the State. Diagnostic tests for HB included serum agglutination, ELISA (IgG or IgM) and polymerase chain reaction (PCR). Clinical, laboratorial and epidemiological data were analyzed. A P value of 0.05 was considered statistically significant. Results: Out of a total of 3,941 patients, 754 presented with a positive test result for HB. After 2014, there was a significant increase in the number of cases, exceeding 100 cases per trimester. In the beginning of 2015, the workgroup of HB started several actions for prevention and treatment, and the number of cases progressively diminished to fewer than 20 cases per trimester. Of 191 reported cases, an occupational risk was found in 84.7%; most cases occurred in farmers (60.0%), veterinarians (17.6%) and slaughterhouse workers (14.7%). Manipulation of animals and unpasteurized milk consumption were associated with positive Brucella IgM ELISA with an odds ratio (OR) of 1.42 (1.09–1.84) and 1.48 (1.01–2.15), respectively. Conclusions: HB outbreaks can occur in low to middle-income countries and are associated with slaughterhouse work, handling of unpasteurized milk and animal manipulation. Intensive programs for control of HB are important to reduce the number of cases. [ABSTRACT FROM AUTHOR]

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Plazomicin is a next-generation aminoglycoside with activity against Enterobacteriaceae , including carbapenemase-producing Enterobacteriaceae (CPE). The aim of this study was to evaluate the activity of plazomicin against CPE ( Klebsiella spp., Escherichia coli , Serratia spp., Enterobacter spp., Citrobacter spp., Morganella spp ., Proteus spp., Providencia spp.) from different Brazilian hospitals. A total of 4000 carbapenem-resistant Enterobacteriaceae isolates were collected from clinical samples in 50 Brazilian hospitals during 2013–2015. Of these, 499 carbapenem-resistant isolates (CLSI criteria) were selected for further evaluation via broth microdilution to assess for the activity of plazomicin, colistin, tigecycline, meropenem, amikacin, and gentamicin. Additionally, the isolates were assessed for the presence of carbapenemase genes ( bla KPC , bla NDM , bla OXA-48-like , bla IMP , bla BKC , bla GES , and bla VIM ) by polymerase chain reaction (PCR). When PCR was positive to bla OXA-48-like , bla IMP , bla GES , and bla VIM, the carbapenemase genes were sequenced. bla KPC was the most prevalent carbapenemase gene found ( n = 397), followed by bla NDM ( n = 81), bla OXA-48 ( n = 12), and bla IMP-1 ( n = 3). Other genes were identified in only 1 isolate each: bla BKC-1 , bla GES-16 , bla GES-1 , bla OXA-370 , and bla VIM-1 . One isolate had 2 carbapenemase genes ( bla KPC and bla NDM ). Thirty-three percent of the isolates were nonsusceptible to colistin, 24% to tigecycline, 97% to meropenem, 51% to amikacin, and 81% to gentamicin (via EUCAST criteria). The plazomicin MIC 50/90 was 0.5/64 mg/L, with 85% of MICs ≤2 mg/L and 87% of MICs ≤4 mg/L. Elevated MICs to plazomicin were not associated with a specific carbapenemase or bacterial species. The MICs of plazomicin against CPE were lower than those of other aminoglycosides. Plazomicin is a promising drug for the treatment of CPE infections. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a major public health problem worldwide. Although next generation sequencing technology has been widely used in the diagnosis of infectious diseases, it has been scarcely applied in identification of Leishmania species. The aim of this study was to compare the efficiency of MinION™ nanopore sequencing and polymerase chain reaction restriction fragment length polymorphism in identifying Leishmania species. Our results showed that the MinION™ sequencer was able to discriminate reference strains and clinical samples with high sensitivity in a cost and time effective manner without the prior need for culture. • The identification of Leishmania species is essential to assist in the proper treatment. • We use the latest generation sequencing for identification of Leishmania species. • In this study we used the third generation MinION sequencer for the first time. • MinION sequencer was able to discriminate reference strains and clinical samples with high sensitivity. [ABSTRACT FROM AUTHOR]

Clostridioides (Clostridium) difficile is the main etiology underlying antibiotic-associated diarrhea (AAD). Still, few Brazilian data are available on this infection. The aims of this multicenter study were to identify the prevalence, clinical characteristics, and outcomes of C. difficile infection (CDI) in patients with antibiotic associated diarrhea at eight hospitals in Curitiba, southern Brazil, during the years 2017–2019. Stool samples were tested using enzyme immunoassay for glutamate dehydrogenase antigen (GDH) and A/B toxins. Positive GDH samples were further evaluated by real-time polymerase chain reaction (PCR) for the presence of genes encoding toxin B (tcdB), binary toxin (cdt), and marker of hypervirulent C. difficile strain (tcdC deletion). The prevalence of CDI in 351 patients with AAD included in the study was 17.7% (n = 62). Among the CDI cases, tcdB was positive in all 62 stool samples, while cdt was positive in 10 samples, and tcdC deletion was positive in only two. Carriage of carbapenem-resistant Gram-negative bacilli, previous hospitalization, and use of broad-spectrum cephalosporin and carbapenem were associated with CDI. Among patients with CDI, 64.5% presented with severe diarrhea, and 8% (5/62) progressed with colitis and required intensive care. The 30-day mortality was 24% (15/62), and the CDI-associated mortality was 4.8% (3/62). Overall, 83.8% (52/62) of the patients achieved primary cure, and 20.8% of the evaluated patients (10/48) presented CDI recurrence. The treatment administered was not significantly associated with the 60-day recurrence or mortality. In conclusion, we reported in this study data of prevalence and recurrence rates of CDI in patients with AAD and evaluated the number of severe cases and infection-related mortality, which were up to now unknown in Southern Brazilian hospitals. Image 1 • Carbapenem resistant Gram-negative bacilli carriage was associate to C. difficile infection. • Use of oral vancomycin or metronidazole was not related to the outcome. • Detection of toxins by EIA was associated with a higher probability of recurrence. [ABSTRACT FROM AUTHOR]