Multiplex PCR to Streptococcus pneumoniae serotype identification directly in cerebrospinal fluid samples.

Streptococcus pneumoniae causes invasive diseases of significant public health concern, such as meningitis. The culture of cerebrospinal fluid (CSF) samples, the standard technique for meningitis diagnoses, is not always positive. Consequently, meaningful information about the etiological agent is lost, which can compromise effective epidemiological surveillance and the improvement of immunization policies. This study aims to standardize a method to genotype pneumococcus in the CSF samples which could mitigate the absence of isolated strains, and also evaluate the prediction of this assay. We applied eight multiplex PCR (mPCR) assays to CSF samples paired with the Quellung reaction applied to the isolated strains. We also compared different master mix kits in the mPCR. Moreover, a retrospective study was conducted with CSF samples considered pneumococcus positive due to the presence of the lytA gene. Results showed that genotyping by the mPCR correlated 100% with the Quellung reaction, and genotyping was dependent on the master mix applied. In the retrospective study (2014-2020), 73.4% were successfully genotyped. The analyses of the receiver operating characteristic curve showed that the cycle threshold (Ct value) around 30 for the lytA gene had a 75% positive chance of successful genotyping, whereas with a Ct value > 35, the chance was 12.5%. Finally, we observed that genotype 19A was prevalent in the period (12%), information unknown until now due to the lack of isolated strains. Therefore, the mPCR of CSF samples can efficiently predict S. pneumoniae serotypes, especially in the absence of isolated strains, which can be a great tool for pneumococcal serotype surveillance.

Tuberculosis/COVID-19 co-infection detected in a single sputum sample using a rapid molecular test.

Tuberculosis (TB) and COVID-19 affect the lungs and are transmitted mainly by aerosols or particles of saliva from infected persons. Clinical similarities between diseases can affect correct diagnosis. Individuals belonging to the population deprived of liberty (PDL) are at increased risk of contagion due to precarious sanitary conditions and overcrowded environments. A variety of specimens may be suitable for the diagnosis of COVID-19, using molecular diagnostic techniques; however, there is little data on the analysis of sputum samples with the Xpert Xpress SARS-CoV-2® for the diagnosis of COVID-19, especially in this population group. The present study reports a case of TB and COVID-19 co-infection detected in sputum from an individual belonging to the PDL. For the detection, it used the GeneXpert platform (Cepheid, USA). Mycobacterium tuberculosis complex (MTC) was detected using the Xpert MTB/RIF Ultra® cartridge and SARS-CoV-2 was detected using the Xpert Xpress SARS-CoV-2® cartridge. The genes IS6110 and IS1081 were detected within 80 min indicating the presence of MTC, with no mutations related to resistance to rifampicin. The SARS-CoV-2 E and N2 genes were detected within 45 min. The result was confirmed by RT-qPCR with detection of E, N, and RdRP/S genes in the sputum and nasopharyngeal (NP) specimens. Rapid diagnoses that allow the identification and differentiation of such diseases are important for adequate epidemiological surveillance, isolation of infected individuals, and interruption of the transmission chain. Using the GeneXpert platform, specimens can be tested as soon as they are received, without the need for prior preparation. The US Food and Drug Administration has issued emergency authorization for the use of the Cepheid Xpert Xpress SARS-CoV-2 for the rapid detection of SARS-CoV-2 using specimens from a NP or nasal wash/aspirate. The case presented here gains an innovation with the use of the sputum to COVID-19 diagnosis.

Alternative SARS-CoV-2 detection protocol from self-collected saliva for mass diagnosis and epidemiological studies in low-incoming regions.

Until mass vaccination befalls, control of the new betacoronavirus-associated severe acute respiratory syndrome pandemic (SARS-CoV-2) is based on decreasing virus circulation by social distancing and blocking transmission foci after diagnosis. Globally adopted SARS-CoV-2 diagnostic criteria embrace viral RNA detection by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) on nasopharynx secretions, which requires healthcare facilities and specialized personnel for sample collection. To develop an alternative protocol, hydrophilic cotton as the material and saliva as the source for biological sample collection in qRT-PCR/RT-endpoint-PCR SARS-CoV-2 diagnostic methods prepared with local consumables were evaluated using 99 archived nasopharynx samples previously diagnosed as positive for SARS-CoV-2 and 111 prospective saliva samples pared with nasopharynx samples from patients attending the local reference ABC Medical School diagnostic laboratory. The kappa agreement coefficient between the SARS-CoV-2 qRT-PCR and RT-endpoint-PCR was k = 0.97 (95 % CI 0.92-1.00) and k = 0.90 (95 % CI 0.81-0.99), respectively, on SARS-CoV-2-positive archived samples, with the initial qRT-PCR CT under 25. The agreement coefficient of the SARS-CoV-2 alternative saliva diagnostic protocol, when used to test the paired nasopharynx samples, was k = 0.79 (95 % CI 0.56-1,00). These data support that the SARS-CoV-2 diagnostic assay based on self-collected saliva on cotton represents an alternative protocol for mass diagnosis and epidemiological studies in low-income regions.

Viability assessment of Mycobacterium tuberculosis complex in OMNIgene • SPUTUM reagent using the BACTEC MGIT 960 System and Xpert MTB/RIF assay.

The World Health Organization advocates that sputum specimens submitted to tuberculosis (TB) diagnostic should be processed within 48 h after collection and be stored under cooling. We aimed to assess the performance of OMNIgene • SPUTUM reagent in maintaining viable specimens of Mycobacterium tuberculosis complex (MTBC) during transportation of sputum samples without refrigeration, in comparison to the standard protocol of the National TB Control Program. Sputum samples obtained in southeastern Brazil (June 2017 to July 2018) from 100 sequential patients with positive acid-fast bacillus smear microscopy were divided into two portions. Portion 1 continued to be cooled (standard protocol, STA), but portion 2 was added to OMNIgene • SPUTUM reagent (alternative protocol, OMS) until concomitant further processing. Both portions of all samples were cultured using MGIT and tested by Xpert MTB/RIF assay. Growth of MTBC in the first 42 days was detected in 96% of the cultures under the STA and 88% under the OMS. Intervals between processing and detecting MTBC growth in the two portions significantly differed (p = 0.0001). Portions under the two protocols showed similar results in the MTBC detection by Xpert assay and culture contamination by non-MTBC. The OMNIgene reagent liquefies and decontaminates sputum leading to a decrease in processing time. Although there was a small delay in mycobacterial growth, the OMNIgene reagent can be useful in specimens transported from collection sites over a long distance to centralized testing centers, maintaining viable MTBC for at least 8 days at room temperature.

Epidemiology and genetic screening of diarrheagenic Escherichia coli among symptomatic and asymptomatic children.

PURPOSE: Enteropathogens are frequently associated with diarrheal disease. Knowledge of their etiology and epidemiology is essential for the prevention and control of the sickness. This study describes the microbiological and epidemiological features of diarrheal disease in 197 symptomatic and 223 asymptomatic under-five-year-old children from southeastern Brazil, between January 2015 and September 2016. METHODS: Isolation of Escherichia coli, Salmonella, Shigella and Campylobacter was realized by culture. E. coli strains were screened by multiplex PCR, PFGE and O:H serotyping. Antimicrobial susceptibility testing was also performed. RESULTS: Most of the 127 enteropathogens isolated were diarrheagenic E. coli (96.1 %), with predominance of several serotypes of enteropathogenic E. coli (EPEC) and enteroaggregative E. coli (EAEC). Age, sex, rotavirus vaccination, recent use of antibiotics and previous contact with pets, were factors that revealed no significant effects on the probability of infection by the predominant pathogens. Even so, higher incomes could be related to a lesser chance of testing positive for EPEC. Evidence of possible EAEC clonal spread was detected, as well as genetic similarity among strains from both symptomatic and asymptomatic children. Resistance to antimicrobial agents was more pronounced among EAEC than EPEC. CONCLUSION: The occurrence of genetically similar diarrheagenic E. coli in both groups of children, likewise resistant to these agents, underscores the importance of establishing strategies for the prevention of outbreaks, especially among low-income households.

Maintenance of demographic and hematological profiles in a long-lasting dengue fever outbreak: implications for management.

BACKGROUND: Dengue fever (DF) outbreaks present regionally specific epidemiological and clinical characteristics. In certain medium-sized cities (100 000-250 000 inhabitants) of São Paulo State, Brazil, and after reaching an incidence of 150 cases/100 000 inhabitants ("epidemiological threshold"), clinical diagnosis indicated dengue virus (DENV) infection. During this period, other seasonally infectious diseases with symptoms and physical signs mimicking DF can simultaneously occur, with the consequential overcrowding of health care facilities as the principal drawbacks. Confirmation of clinical diagnosis of DF with serological tests may help in avoiding faulty diagnosis in patients, who might later undergo dengue hemorrhagic fever (DHF) and the dengue-shock syndrome (DSS). Furthermore, demographic and hematological profiles of patients are useful in detecting specific early characteristics associated to DF, DHF and DSS. METHODS: From March to June, 2007, 456 patients from Marilia in northwest São Paulo State who had only been diagnosed for DF by clinical criteria, underwent serologic testing for non-structural 1 (NS1) DENV antigens. Individual results were used in comparative analysis according to demographic (gender, age) and hematological (leukocyte and platelet counts, percentage of atypical lymphocytes) profiles. Temporal patterns were evaluated by subdividing data according to time of initial attendance, using recorded variables as predictors of DENV infection in logistic regression models and ROC curves. RESULTS: Serologic DENV detection was positive in 70.6 % of the patients. Lower leukocyte and platelet counts were the most important factors in predicting DENV infection (respective medians DENV + = 3 715 cells/ml and DENV- = 6 760 cells/ml, and DENV + = 134 896 cells/ml and DENV- = 223 872 cells/ml). Furthermore, all demographic and hematological profiles presented a conservative temporal pattern throughout this long-lasting outbreak. CONCLUSIONS: As consistency throughout the epidemic facilitated defining the conservation pattern throughout the early stages, this was useful for improving management during the remaining period.