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Background: Despite widespread vaccination, pertussis has re-emerged as a serious public health concern worldwide. Since 2017, Peru has experienced an increase in pertussis cases exhibiting a higher risk of severity and death in young infants. Thus, a dose of the tetanus, diphtheria, and acellular pertussis (Tdap) vaccine is recommended for pregnant women in the third trimester. Although evidence suggests the maternal Tdap vaccine is safe and effective, its association with a reduced risk of pertussis in developing countries remains poorly investigated. Methods: We conducted a case-control study to evaluate the association between Tdap vaccination during pregnancy and reduction in the risk of pertussis among infants aged <2 months in Peru. Pertussis cases and controls treated in healthcare facilities nationwide between 2019 and 2021 and confirmed by real-time polymerase chain reaction were included. The controls were randomly selected from test-negative patients. Odds ratios (ORs) and vaccine effectiveness (VE) were calculated using a multiple logistic regression model and 1 - (OR) × 100%, respectively. Results: Fifty cases and 150 controls were included in the analysis. The mothers of 4% of cases and 16.7% of controls received Tdap vaccination during pregnancy, resulting in an OR of 0.19 (95% confidence interval [CI], .04-.86) and VE of 81% (95% CI, 14%-96%) for preventing pertussis in infants. Conclusions: Peruvian infants <2 months old whose mothers received the Tdap vaccine in the third trimester of pregnancy had a significantly lower risk of pertussis. The Tdap vaccination is thus an effective intervention to reduce the burden of pertussis in at-risk populations.

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Despite widespread vaccination, Bordetella pertussis continues to cause pertussis infections worldwide, leaving infants at the highest risk of severe illness and death, while people around them are likely the main sources of infection and rapidly spread the disease. Rapid and less complex molecular testing for the specific and timely diagnosis of pertussis remains a challenge that could help to prevent the disease from worsening and prevent its transmission. We aimed to develop and validate a colorimetric loop-mediated isothermal amplification (LAMP) assay using a new target uvrD_2 informed by the pangenome for the specific and early detection of B. pertussis. Compared to that of multitarget quantitative polymerase chain reaction (multitarget qPCR) using a large clinical DNA specimen (n = 600), the diagnostic sensitivity and specificity of the uvrD_2 LAMP assay were 100.0% and 98.6%, respectively, with a 99.7% degree of agreement between the two assays. The novel colorimetric uvrD_2 LAMP assay is highly sensitive and specific for detecting B. pertussis DNA in nasopharyngeal swabs and showed similar diagnostic accuracy to complex and high-cost multitarget qPCR, but it is faster, simpler, and inexpensive, which makes it very helpful for the reliable and timely diagnosis of pertussis in primary health care and resource-limited settings.

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OBJECTIVE.: To develop and evaluate a low-cost cellulose-based method for rapid purification and direct amplification of Bordetella pertussis DNA from nasopharyngeal swabs. MATERIALS AND METHODS.: We prepared cellulose discs and evaluated different parameters (lysis/wash buffers, number of discs and DNA elution). The method was coupled to a direct real-time PCR (qPCR) amplification and the performance was estimated using nasopharyngeal swabs that were positive (n=100) and negative (n=50) for B. pertussis DNA by qPCR, compared to the silica column-based method. We calculated sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) and the degree of agreement. The feasibility of the rapid method to be coupled to a loop-mediated isothermal amplification colorimetric assay (LAMP) was evaluated. RESULTS.: The rapid method, with a cellulose disk and lysis and wash buffer containing PVP-40 and Tween 20, respectively, showed a greater capacity to purify amplifiable DNA from B. pertussis. The method had a sensitivity of 89.0% (95%CI: 80.2%-94.9%) and a specificity of 98.5% (95%CI: 92.1%-100.0%), with a good degree of agreement (Kappa=0.867; 95%CI: 0.788 - 0.946), compared to the reference method. The PPV and NPV were 98.6% (95%CI: 92.7.2%-100.0%) and 88.2% (95%CI: 78.7%-94.4%), respectively. Successful amplification by LAMP was evident, and comparable results were obtained with the silica column method. CONCLUSION.: The developed method is simple, low-cost and equipment-free for rapid (60 seconds) DNA collection at the point of care, and can be implemented in various molecular techniques aimed at the timely diagnosis and epidemiological study of pertussis.

OBJETIVO.: Desarrollar y evaluar un método de bajo costo basado en celulosa para la purificación rápida y amplificación directa de ADN de Bordetella pertussis de hisopados nasofaríngeos. MATERIALES Y MÉTODOS.: Se prepararon discos de celulosa y se evaluaron diferentes parámetros (buffers de lisis/lavado, número de discos y elución de ADN). El método se acopló a una amplificación directa por PCR en tiempo real (qPCR) y se estimó el rendimiento utilizando hisopados nasofaríngeos que fueron positivos (n=100) y negativos (n=50) para ADN B. pertussis por qPCR, comparado con el método basado en columnas de sílice. Se calculó el grado de concordancia, sensibilidad, especificidad, valor predictivo positivo (VPP) y valor predictivo negativo (VPN). Se evaluó la factibilidad del método rápido para ser acoplado a un ensayo colorimétrico de amplificación isotérmica mediada por lazo (LAMP). RESULTADOS.: El método rápido con un disco de celulosa y buffer de lisis y lavado conteniendo PVP-40 y Tween 20, respectivamente, mostró una mayor capacidad para purificar ADN amplificable de B. pertussis. El método tuvo una sensibilidad de 89,0% (IC95%, 80,2%-94,9%) y una especificidad de 98,5% (IC95%, 92,1%-100,0%), con un buen grado de concordancia (Kappa=0,867; IC95% 0,788 - 0,946), respecto al método referencial. Los VPP y VPN fueron 98,6% (IC95%, 92,7,2%-100,0%) y 88,2% (IC95%, 78,7%-94,4%), respectivamente. Se evidenció una amplificación exitosa por LAMP, y se obtuvieron resultados comparables con el método por columnas de sílice. CONCLUSIÓN.: El método desarrollado es simple, de bajo costo y libre de equipos para la obtención rápida (60 segundos) de ADN en el punto de atención, y puede ser implementado en diversas técnicas moleculares orientados al diagnóstico oportuno y al estudio epidemiológico de tos ferina.

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RESUMEN Objetivo. Desarrollar y evaluar un método de bajo costo basado en celulosa para la purificación rápida y amplificación directa de ADN de Bordetella pertussis de hisopados nasofaríngeos. Materiales y métodos. Se prepararon discos de celulosa y se evaluaron diferentes parámetros (buffers de lisis/lavado, número de discos y elución de ADN). El método se acopló a una amplificación directa por PCR en tiempo real (qPCR) y se estimó el rendimiento utilizando hisopados nasofaríngeos que fueron positivos (n=100) y negativos (n=50) para ADN B. pertussis por qPCR, comparado con el método basado en columnas de sílice. Se calculó el grado de concordancia, sensibilidad, especificidad, valor predictivo positivo (VPP) y valor predictivo negativo (VPN). Se evaluó la factibilidad del método rápido para ser acoplado a un ensayo colorimétrico de amplificación isotérmica mediada por lazo (LAMP). Resultados. El método rápido con un disco de celulosa y buffer de lisis y lavado conteniendo PVP-40 y Tween 20, respectivamente, mostró una mayor capacidad para purificar ADN amplificable de B. pertussis. El método tuvo una sensibilidad de 89,0% (IC95%, 80,2%-94,9%) y una especificidad de 98,5% (IC95%, 92,1%-100,0%), con un buen grado de concordancia (Kappa=0,867; IC95% 0,788 - 0,946), respecto al método referencial. Los VPP y VPN fueron 98,6% (IC95%, 92,7,2%-100,0%) y 88,2% (IC95%, 78,7%-94,4%), respectivamente. Se evidenció una amplificación exitosa por LAMP, y se obtuvieron resultados comparables con el método por columnas de sílice. Conclusión. El método desarrollado es simple, de bajo costo y libre de equipos para la obtención rápida (60 segundos) de ADN en el punto de atención, y puede ser implementado en diversas técnicas moleculares orientados al diagnóstico oportuno y al estudio epidemiológico de tos ferina.

ABSTRACT Objective. To develop and evaluate a low-cost cellulose-based method for rapid purification and direct amplification of Bordetella pertussis DNA from nasopharyngeal swabs. Materials and methods. We prepared cellulose discs and evaluated different parameters (lysis/wash buffers, number of discs and DNA elution). The method was coupled to a direct real-time PCR (qPCR) amplification and the performance was estimated using nasopharyngeal swabs that were positive (n=100) and negative (n=50) for B. pertussis DNA by qPCR, compared to the silica column-based method. We calculated sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) and the degree of agreement. The feasibility of the rapid method to be coupled to a loop-mediated isothermal amplification colorimetric assay (LAMP) was evaluated. Results. The rapid method, with a cellulose disk and lysis and wash buffer containing PVP-40 and Tween 20, respectively, showed a greater capacity to purify amplifiable DNA from B. pertussis. The method had a sensitivity of 89.0% (95%CI: 80.2%-94.9%) and a specificity of 98.5% (95%CI: 92.1%-100.0%), with a good degree of agreement (Kappa=0.867; 95%CI: 0.788 - 0.946), compared to the reference method. The PPV and NPV were 98.6% (95%CI: 92.7.2%-100.0%) and 88.2% (95%CI: 78.7%-94.4%), respectively. Successful amplification by LAMP was evident, and comparable results were obtained with the silica column method. Conclusion. The developed method is simple, low-cost and equipment-free for rapid (60 seconds) DNA collection at the point of care, and can be implemented in various molecular techniques aimed at the timely diagnosis and epidemiological study of pertussis.

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PURPOSE: The purpose of this study is to evaluate the frequency of viral and bacterial respiratory pathogens detected by molecular methods in sputum samples of patients hospitalized for COVID-19 and to evaluate its impact on mortality and unfavorable outcomes (in-hospital death or mechanical ventilation). PATIENTS AND METHODS: The prospective cohort included patients with diagnosis of COVID-19 hospitalized at Hospital Nacional Hipólito Unanue. Sociodemographic and clinical data were collected from clinical records. Sputum samples were analyzed with the Biofire Filmarray Pneumonia plus® respiratory panel. Crude and adjusted associations with unfavorable outcomes were evaluated using logistic regression models. RESULTS: Ninety-three patients who were able to collect sputum samples were recruited between September 8 and December 28, 2020. The median age was 61.7 years (IQR 52.3-69-8) and 66 (71%) were male. The most frequent symptoms were dyspnea, cough, fever, and general malaise found in 80 (86%), 76 (82%), 45 (48%), and 34 (37%) patients, respectively. Fifty-three percent of patients had comorbidities. Seventy-six (82%) patients received antibiotics prior to admission and 29 (31%) developed unfavorable outcome. Coinfection was evidenced in 38 (40.86%) cases. The most frequently found bacteria were Staphylococcus aureus, Streptococcus agalactiae, Haemophilus influenzae and Klebsiella pneumoniae in 11 (11.83%), 10 (10.75%), 10 (10.75%), and 8 (8.6%) cases, respectively. Streptococcus pneumoniae was found in one case (1.08%). We neither identify atypical bacteria nor influenza virus. No association was found between the presence of viral or bacterial microorganisms and development of unfavorable outcomes (OR 1.63; 95% CI 0.45-5.82). CONCLUSION: A high frequency of respiratory pathogens was detected by molecular methods in patients with COVID-19 pneumonia but were not associated with unfavorable outcomes. No atypical agents or influenza virus were found. The high use antibiotics before admission is a concern. Our data suggest that the use of drug therapy against atypical bacteria and viruses would not be justified in patients hospitalized for COVID-19.

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Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a major threat to public health. Rapid molecular testing for convenient and timely diagnosis of SARS-CoV-2 infections represents a challenge that could help to control the current pandemic and prevent future outbreaks. We aimed to develop and validate a multiplex and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using lyophilized LAMP reagents for sensitive and rapid detection of SARS-CoV-2. LAMP primers were designed for a set of gene targets identified by a genome-wide comparison of viruses. Primer sets that showed optimal features were combined into a multiplex RT-LAMP assay. Analytical validation included assessment of the limit of detection (LoD), intra- and inter-assay precision, and cross-reaction with other respiratory pathogens. Clinical performance compared to that of real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) was assessed using 278 clinical RNA samples isolated from swabs collected from individuals tested for COVID-19. The RT-LAMP assay targeting the RNA-dependent RNA polymerase (RdRp), membrane (M), and ORF1ab genes achieved a comparable LoD (0.65 PFU/mL, CT=34.12) to RT-qPCR and was 10-fold more sensitive than RT-qPCR at detecting viral RNA in clinical samples. Cross-reactivity to other respiratory pathogens was not observed. The multiplex RT-LAMP assay demonstrated a strong robustness and acceptable intra- and inter-assay precision (mean coefficient of variation, 4.75% and 8.30%). Diagnostic sensitivity and specificity values were 100.0% (95% CI: 97.4-100.0%) and 98.6% (95% CI: 94.9-99.8%), respectively, showing high consistency (Cohen's kappa, 0.986; 95% CI: 0.966-1.000; p<0.0001) compared to RT-qPCR. The novel one-step multiplex RT-LAMP assay is storable at room temperature and showed similar diagnostic accuracy to conventional RT-qPCR, while being faster (<45 min), simpler, and cheaper. The new assay could allow reliable and early diagnosis of SARS-CoV-2 infections in primary health care. It may aid large-scale testing in resource-limited settings, especially if it is integrated into a point-of-care diagnostic device.

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Peru has become one of the countries with the highest mortality rates from the current coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To investigate early transmission events and the genomic diversity of SARS-CoV-2 isolates circulating in Peru in the early COVID-19 pandemic, we analyzed 3472 viral genomes, of which 149 were from Peru. Phylogenomic analysis revealed multiple and independent introductions of the virus likely from Europe and Asia and a high diversity of genetic lineages circulating in Peru. In addition, we found evidence for community-driven transmission of SARS-CoV-2 as suggested by clusters of related viruses found in patients living in different regions of Peru.

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Asymptomatic carriers are a likely source of transmission of Neisseria meningitidis to close contacts who are placed at a higher risk for invasive meningococcal disease (IMD). Although N. meningitidis ciprofloxacin-resistance is rare, there have been an increase in the reports of resistant isolates mainly in patients diagnosed with IMD, and little is known about the N. meningitidis ciprofloxacin-resistance in the carrier populations. We performed a pharyngeal carriage study during a 2017 military setting outbreak in Peru, caused by a ciprofloxacin-resistant N. meningitidis B. The isolates analysed came from two hospitalized cases and six asymptomatic carriers. Whole-genome sequence-based analysis was performed and showed that strains carrying the Thr91Ile mutation, in the gene encoding for subunit A of DNA gyrase (gyrA), were responsible for the fluoroquinolone resistance (MICs ≥0.256 µg ml-1) and were closely related to highly virulent strains from France, Norway and the UK. Phylogenetic analysis of the gyrA gene revealed that likely these Peruvian isolates acquired resistance through horizontal gene transfer from Neisseria lactamica. Our study provides evidence for the emergence and propagation of ciprofloxacin-resistant N. meningitidis B from asymptomatic carriers, and recommends the introduction of serogroup B vaccines for high-risk populations.

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