Development of a national proficiency test for SARS-CoV-2 detection by PCR in Colombia.

Background: Proficiency testing (PT) is a tool for ensuring the validity of results of testing laboratories and is essential when laboratories are working with assays authorised for emergency use or implementing novel techniques for detecting emerging pathogens. Methods: In collaboration with the National Health Institute of Colombia and with international support, we developed a qualitative PT for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by reverse transcription polymerase chain reaction (RT-PCR). A proficiency test item (PTI) based on reference material (research grade) produced by the National Institute of Standards and Technologies (NIST) was prepared and characterised using three positive samples with varying concentrations of SARS-CoV-2 ribonucleic acid (RNA) and two negative (control) samples. Tests were distributed to 121 laboratories across the national network of public health laboratories in Colombia. Results: Positive samples had varying concentrations of SARS-CoV-2 RNA and were quantified by digital PCR (RT-ddPCR) assays for the E gene of SARS-CoV-2. We tested the ability of laboratories to detect low and high levels of viral RNA using samples with SARS-CoV-2 RNA concentrations of 1417 ± 216, 146 ± 28, and 14 ± 10 copies /uL (expanded uncertainty, k = 2, 95% confidence level) We also performed a semiquantitative analysis of instrumental responses (Ct values) reported by participating laboratories and homogeneity, stability, and characterisation studies of the produced materials to determine the adequacy of these materials and methods for use in the qualitative PT scheme. The PT evaluated reports for individual target genes from each laboratory; 98.3% of laboratories had satisfactory performance and the remaining 1.7% of laboratories had unsatisfactory performance for the detection of at least one of the reported genes. Conclusions: This PT scheme identified the potential metrological weaknesses of laboratories in the detection of SARS-CoV-2 by RT-PCR and may facilitate improvements in the quality of measurements from the perspective of public health surveillance.

Comprehensive Automated Routine Implementation, Validation, and Benchmark of the Anisotropic Force Field (AUA4) Using Python and GROMACS.

Molecular simulation users are sometimes discouraged from using specific molecular models because of the inconvenience of finding the force field parameters and preparing and validating the topology files. To facilitate this process and make the accurate anisotropic force field AUA4 available to molecular dynamics users, we have created and validated an automated topology and coordinate file creation routine for the GROMACS molecular simulation software. In the present work, we describe the AUA4, explain its particularities and how it was implemented, thoroughly validating the implementation, and for the first time, perform a molecular dynamics benchmark for this transferable force field. Several properties were computed, namely, liquid density, vapor pressure, and vaporization enthalpy by conducting explicit vapor-liquid interface simulations. The results evidence the correct implementation showing slight deviations from the parametrization studies. The benchmark shows the superior predictive capability of the AUA4 in recreating liquid density (RMSD equal to 17.0 kg/m3) and vaporization enthalpy (RMSD equal to 1.3 kJ/mol) compared to other transferable force fields. In addition, its superior computational time performance doubles or even triples compared to an all-atom force field such as the OPLS, depending on whether the workstation counts with GPU.

Anticuerpos neutralizantes contra el virus de la fiebre amarilla 17 D en colombianos vacunados y no vacunados con inmunidad a dengue / Yellow fever virus 17D neutralising antibodies in vaccinated Colombian people and unvaccinated ones having immunity against dengue

Objetivo Determinar la frecuencia de título protector de anticuerpos neutralizantes contra el virus de la fiebre amarilla (AN-VFA a título >1:10) en colombianos vacunados con la cepa 17 D y conocer la magnitud de neutralización del VFA por anticuerpos contra dengue. Metodología Se colectó suero de 100 individuos con vacuna documentada por carné y de 116 residentes en municipios de Norte de Santander afectados por el brote en 2002-2003, quienes informaron haber sido vacunados. Se incluyeron sueros de individuos no vacunados con (n=61) y sin (n=16) anticuerpos contra dengue. Todos los sueros se analizaron por la prueba de neutralización para VFA por 75 por ciento de reducción de placa. Resultados AN-VFA a título >1:10 se encontraron en 90 por ciento de vacunados con carné y sin variación aparente en relación con edad. Al contrario, hubo correlación entre disminución de la frecuencia de título protector de anticuerpos e incremento del tiempo de inmunización (r=0,95; p=0,04). En residentes de Norte de Santander, AN-VFA a título >1:10 se encontraron en 92,6 por ciento adultos y 69 por ciento niños. El VFA fue neutralizado (52 -100 por ciento) por sueros de inmunes a dengue más eficientemente que por sueros de no inmunes (p<0.001). Conclusiones Vacunados con el virus 17 D podrían no estar protegidos contra fiebre amarilla: hasta 31 por ciento niños y 10 por ciento adultos. Anticuerpos contra dengue inhibieron el VFA y su significancia en términos de protección contra fiebre amarilla deberá ser investigada.

Objective Determining the frequency of yellow fever seroprotective antibody neutralising titres (YF-NT >1:10) in Colombians vaccinated with the 17 D virus and ascertaining the extent to which YF virus can be neutralised by dengue antibodies. Materials and Methods Serum samples were taken from 100 subjects who showed their vaccination record and from 116 residents in municipalities (Norte de Santander) affected by a wild YF outbreak in 2002-2003 who were reported to have been YF vaccinated. Sera from individuals with (n=61) and without (n=16) dengue antibodies who had never been YF vaccinated were included. All the sera were tested by 75 percent YF plaque-reduction neutralization test. Results YF-NT titres >1:10 were founded in 90 percent of subjects with vaccination recorded with minors variations in relation to age. In contrast, there was correlation between decrease of seroprotective YF-NT titres frequency and increase of immunization time (r=0.95; p=0.04). In residents in YF endemic area, YF-NT titres > 1.10 were founded in 92,6 percent adults and 69 percent children. YF 17 D virus was neutralized (52-100 percent) by dengue sera more efficiently than non-dengue immune sera (p<0.001). Conclusions Individuals immunised with YF vaccine 17 D could not be protected against YF: up to 31 percent children and 10 percent adults. Dengue antibodies inhibited YF virus and its significance in terms of YF protection must be investigated.

[Yellow fever virus 17D neutralising antibodies in vaccinated Colombian people and unvaccinated ones having immunity against dengue]. / Anticuerpos neutralizantes contra el virus de la fiebre amarilla 17 D en colombianos vacunados y no vacunados con inmunidad a dengue.

OBJECTIVE: Determining the frequency of yellow fever seroprotective antibody neutralising titres (YF-NT >or=1:10) in Colombians vaccinated with the 17 D virus and ascertaining the extent to which YF virus can be neutralised by dengue antibodies. MATERIALS AND METHODS: Serum samples were taken from 100 subjects who showed their vaccination record and from 116 residents in municipalities (Norte de Santander) affected by a wild YF outbreak in 2002-2003 who were reported to have been YF vaccinated. Sera from individuals with (n=61) and without (n=16) dengue antibodies who had never been YF vaccinated were included. All the sera were tested by 75 % YF plaque-reduction neutralization test. RESULTS: YF-NT titres >or=1:10 were founded in 90 % of subjects with vaccination recorded with minors variations in relation to age. In contrast, there was correlation between decrease of seroprotective YF-NT titres frequency and increase of immunization time (r=0.95; p=0.04). In residents in YF endemic area, YF-NT titres >or= 1.10 were founded in 92,6 % adults and 69 % children. YF 17 D virus was neutralized (52-100 %) by dengue sera more efficiently than non-dengue immune sera (p<0.001). CONCLUSIONS: Individuals immunised with YF vaccine 17 D could not be protected against YF: up to 31% children and 10 % adults. Dengue antibodies inhibited YF virus and its significance in terms of YF protection must be investigated.

[Dengue hemorrhagic fever serotype and infection pattern in a Colombian endemic area]. / Serotipo, Patrón de Infección y Dengue Hemorrágico en Area Endémica Colombiana.

OBJECTIVE: Describing the relationship between viral serotypes, infection pattern and dengue hemorrhagic fever. METHODS: 1,545 febrile patients were studied from 1998-2004 in the Santander department of Colombia. Dengue infection was confirmed by IgM ELISA and the virus was isolated in C6/36 cells. Infection pattern was established by detecting IgG antibodies in acute serum. Neutralising antibody titres were investigated in dengue cases occurring during years when less (1998) and more (2001) dengue hemorrhagic cases were reported by using PRNT. RESULTS: DEN-1 predominance in 1998 and the re-introduction of DEN-3 in 2001 coincided with an epidemic. DEN-2 infection caused more hemorrhagic cases than DEN-3 infection (24,5 % cf 11,2 %; p<0.05). DEN-2 was more associated with secondary infection than DEN-3 (56,8 % cf 15,7 %; p<0.001). An annual decrease of DHF was correlated with decreased DEN-2 dominance (r=0.95; p= 0.01), and secondary infection (r=0.9; p=0.03) and increased DEN-3 predominance (r=-0.91; p=0.03). There were no differences in neutralising antibody titres amongst analysed cases. DEN-1 neutralising antibodies presented the highest titres. CONCLUSIONS: Change in relative dengue virus serotype abundance was associated with changed infection pattern and DHF frequency. Continuing virological surveillance should become a priority for preventing dengue hemorrhagic fever in endemic areas.

Serotipo, Patrón de Infección y Dengue Hemorrágico en Area Endémica Colombiana / Dengue hemorrhagic fever serotype and infection pattern in a Colombian endemic area

Objetivo: La relación entre serotipo del virus, patrón de infección y dengue hemorrágico es presentada. Métodos: Se estudiaron 1 545 pacientes febriles de municipios del Departamento de Santander, Colombia, entre 1998-2004. El dengue se confirmó por ELISA-IgM y el aislamiento viral se hizo en células C6/36. El patrón de infección se estableció investigando anticuerpos IgG en suero agudo. El título de anticuerpos neutralizantes se determinó usando la prueba de neutralización por reducción de placa (PRNT). Resultados: Predominancia del DEN-1 en 1998 y re-introducción del DEN-3 en 2001 coincidieron con epidemias. El dengue hemorrágico fue más frecuente en infecciones por virus DEN-2 que DEN-3 (24,5 por ciento vs 11,2 por ciento; p<0,05). El DEN-2 se asoció más con infección secundaria que el DEN-3 (56,8 por ciento vs 15,7 por ciento; p< 0,001). Disminución anual del DH correlacionó con disminución de la dominancia del DEN-2 (r = 0,95, p=0,01) y de la infección secundaria (r=0,9; p=0,03) e incremento de la dominancia del DEN-3 (r=-0,91; p=0,03). No se encontraron diferencias en el título de anticuerpos neutralizantes en los casos analizados. Los anticuerpos neutralizantes del DEN-1 fueron los de mayor título. Conclusión: Cambios en la abundancia relativa de serotipos del virus se asociaron con cambios en el patrón de infección y frecuencia del dengue hemorrágico. La vigilancia virológica permanente deberá ser prioridad para la prevención del dengue hemorrágico en áreas endémicas.

Objective: Describing the relationship between viral serotypes, infection pattern and dengue hemorrhagic fever. Methods: 1 545 febrile patients were studied from 1998-2004 in the Santander department of Colombia. Dengue infection was confirmed by IgM ELISA and the virus was isolated in C6/36 cells. Infection pattern was established by detecting IgG antibodies in acute serum. Neutralising antibody titres were investigated in dengue cases occurring during years when less (1998) and more (2001) dengue hemorrhagic cases were reported by using PRNT. Results: DEN-1 predominance in 1998 and the re-introduction of DEN-3 in 2001 coincided with an epidemic. DEN-2 infection caused more hemorrhagic cases than DEN-3 infection (24,5 percent cf 11,2 percent; p<0.05). DEN-2 was more associated with secondary infection than DEN-3 (56,8 percent cf 15,7 percent; p<0.001). An annual decrease of DHF was correlated with decreased DEN-2 dominance (r=0.95; p= 0.01), and secondary infection (r=0.9; p=0.03) and increased DEN-3 predominance (r=-0.91; p=0.03). There were no differences in neutralising antibody titres amongst analysed cases. DEN-1 neutralising antibodies presented the highest titres. Conclusions: Change in relative dengue virus serotype abundance was associated with changed infection pattern and DHF frequency. Continuing virological surveillance should become a priority for preventing dengue hemorrhagic fever in endemic areas.

[Subtypes of dengue virus serotypes 2, 3 and 4 isolated in Santander District, Colombia]. / Subtipos de virus dengue serotipos 2, 3 y 4 aislados en el Departamento de Santander, Colombia.

Virus serotypes 2, 3 and 4 that had circulated in Santander District, Colombia in the period 1998-2004 were analyzed. Identifying the subtype of a dengue virus serotype is a useful tool for surveillance of severe risk factors because the strain potential to cause hemorrhagic dengue makes the difference among them. Simultaneous sequence amplification technique known as restriction site specific-polymerase chain reaction (RSS-PCR) was used to determine the subtype by comparing the electrophoretic pattern of the local isolate to the reference virus. Virus serotype 2 corresponded to subtype A similar to the one isolated in Thailand (1996) and to the other isolated in Porto Rico (1986); virus serotypes 3 were of subtype C like the virus found in Sri Lanka (1990), Honduras (1995) and Porto Rico (2000); virus serotypes 4 were a variant of subtype B similar to a virus from Porto Rico (1987) and to another virus from Tahiti (1985). The study confirmed the presence in Colombia of dengue virus subtypes circulating now in the Americas.