Development of an amplicon-based sequencing approach in response to the global emergence of mpox.

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.

Development of an amplicon-based sequencing approach in response to the global emergence of human monkeypox virus.

The 2022 multi-country monkeypox (mpox) outbreak concurrent with the ongoing COVID-19 pandemic has further highlighted the need for genomic surveillance and rapid pathogen whole genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for SARS-CoV-2. Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical samples that tested presumptive positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR cycle threshold below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.

Citologías cervico-vaginales no satisfactorias en el hospital “Manuel Noriega Trigo” de Maracaibo / Unsatisfactory cervico-pap tests at the hospital

Objetivo: Determinar el porcentaje de citologías cervico-vaginales no satisfactorias en el Hospital “Manuel Noriega Trigo”. Métodos: Se revisaron retrospectivamente las citologías cervico-vaginales estudiadas en el Servicio de Anatomía-Patológica del período comprendido entre noviembre 2008 a diciembre de 2009. Resultados: Se revisaron 1 566 citologías cervico-vaginales reportándose 126 como insatisfactorias (8.%). Las causas más frecuente de citologías cervico-vaginales no satisfactoria fue la falta de identificación del paciente (n=53, 36,8 %), la abundancia de leucocitos (n=31, 21,5 %) y la presencia de abundantes hematíes (n=26, 18,1 %). Diez y siete citologías cervico-vaginales (14,3 %) presentaron varias causas para ser catalogadas como insatisfactorias. Ochenta (63,5 %) de 126 citologías cervico-vaginales reportadas insatisfactorias fueron reportadas como negativas para malignidad. Cuarenta (31,7 %) de las (126 citologías cervico-vaginales) insatisfactorias fueron consideradas como imposibles de ser analizadas o estudiadas por el citotecnólogo o patólogo, representaron el 2,6 % del total de las estudiadas. En 33 citologías cervico-vaginales (2,1 %), no se encontraron células endocervicales. Los centros asistenciales que presentaron un mayor número de citologías cervico-vaginales no satisfactorias fueron el Hospital “Manuel Noriega Trigo” con 63 (50 %), Ambulatorio El Silencio con 31 (24,6 %) y el Ambulatorio El Caujaro con 17 (13,5 %), sin embargo, en proporción, fueron los Ambulatorios El Silencio (31 de 133 citologías cervico-vaginales, 23,3 %) y El Caujaro (17 de 115 citologías cervico-vaginales, 14,8 %) los que tuvieron mayor número de citologías cervico-vaginales insatisfactorias. Conclusiones: El porcentaje de citologías cervico-vaginales insatisfactorias fue aceptable cuando se analizaron las causas propias de la toma, procesamiento y lecturas de las citologías cervico-vaginales. Asimismo, el porcentaje de ausencia de células ...

Objective: To determinate the percentage of unsatisfactory Pap smears at Manuel Noriega Trigo Hospital. Methods: Pap smears studied by the Service of Pathology were reviewed retrospectively between November 2008 and December 2009. Results: One hundred twenty six (8 %) of 1 566 Pap smears were reported unsatisfactory. The most common cause of unsatisfactory vaginal cytology was the lack of patient´s identification (n=53, 36.8 %), obscuring smear inflammation (n=31, 21.5 %) and blood (n=26, 18.1 %). Seventeen Pap smear (14.3 %) had several causes to be considered non satisfactory. Eighty (63.5 %) of 126 Pap smear unsatisfactory were negatives to malignancy. Forty (31.7 %) of 126 Pap smear unsatisfactory could not be analyzed neither the cytotechnogist nor the pathologist; they represented 2.6 % of the 1566 Pap smear. Thirty three Pap smear (2.1 %) did not have endocervical cells. Hospital “Manuel Noriega Trigo” (n=63.50 %), El Silencio (n=31, 24.6 %) and El Caujaro (n=17, 13.5 %) have higher number of unsatisfactory vaginal cytologies, however, proportionally El Silencio (31 of 133 Pap smear, 23.3 %) and El Caujaro (17 of 115 Pap smear, 14.8 %) had higher unsatisfactory Pap smears. Conclusion: The percentage of unsatisfactory Pap smears was acceptable when we analyzed causes such as the technique of collection of the Pap smear, the staining technique, and the study of the slide. The number of endocervical cells was low even though the endocervical collection sample was taken with a cotton swab, maybe because Pap smear were taken by medical doctors.