• View in gallery
    Figure 1.

    Correlation of whole blood specimens paired with dried blood spots by qPCR for Plasmodium spp. Line of best fit (y = 1.02x + 5.41, R2 = 0.889) of Ct-values, excluding negative values (Ct = 40). Ct = cycle threshold; DBS = dried blood spot; qPCR = quantitative polymerase chain reaction.

  • 1.

    Iroh Tam PY , Obaro SK , Storch G , 2016. Challenges in the etiology and diagnosis of acute febrile illness in children in low- and middle-income countries. J Pediatric Infect Dis Soc 5: 190205.

    • Search Google Scholar
    • Export Citation
  • 2.

    Prasad N , Murdoch DR , Reyburn H , Crump JA , 2015. Etiology of severe febrile illness in low- and middle-income countries: a systematic review. PLoS One 10: e0127962.

    • Search Google Scholar
    • Export Citation
  • 3.

    Liu J et al.2016. Development of a TaqMan Array Card for acute-febrile-illness outbreak investigation and surveillance of emerging pathogens, including ebola virus. J Clin Microbiol 54: 4958.

    • Search Google Scholar
    • Export Citation
  • 4.

    Sutcliffe CG , Mutanga JN , Moyo N , Schue JL , Hamahuwa M , Thuma PE , Moss WJ , 2020. Acceptability and feasibility of testing for HIV infection at birth and linkage to care in rural and urban Zambia: a cross-sectional study. BMC Infect Dis 20: 227.

    • Search Google Scholar
    • Export Citation
  • 5.

    Nnodu OE et al.2020. Implementing newborn screening for sickle cell disease as part of immunisation programmes in Nigeria: a feasibility study. Lancet Haematol 7: e534e540.

    • Search Google Scholar
    • Export Citation
  • 6.

    Gupta K , Mahajan R , 2018. Applications and diagnostic potential of dried blood spots. Int J Appl Basic Med Res 8: 12.

  • 7.

    Canier L et al.2015. Malaria PCR detection in Cambodian low-transmission settings: dried blood spots versus venous blood samples. Am J Trop Med Hyg 92: 573577.

    • Search Google Scholar
    • Export Citation
  • 8.

    Zainabadi K , Adams M , Han ZY , Lwin HW , Han KT , Ouattara A , Thura S , Plowe CV , Nyunt MM , 2017. A novel method for extracting nucleic acids from dried blood spots for ultrasensitive detection of low-density Plasmodium falciparum and Plasmodium vivax infections. Malar J 16: 377.

    • Search Google Scholar
    • Export Citation
  • 9.

    Aubry M et al.2012. Use of serum and blood samples on filter paper to improve the surveillance of dengue in Pacific Island countries. J Clin Virol 55: 2329.

    • Search Google Scholar
    • Export Citation
  • 10.

    Musso D , Roche C , Marfel M , Bel M , Nilles EJ , Cao-Lormeau VM , 2014. Improvement of leptospirosis surveillance in remote Pacific Islands using serum spotted on filter paper. Int J Infect Dis 20: 7476.

    • Search Google Scholar
    • Export Citation
  • 11.

    Selva L et al.2013. Detection of Streptococcus pneumoniae and Haemophilus influenzae type B by real-time PCR from dried blood spot samples among children with pneumonia: a useful approach for developing countries. PLoS One 8: e76970.

    • Search Google Scholar
    • Export Citation
  • 12.

    von Kalckreuth V et al.2016. The Typhoid Fever Surveillance in Africa Program (TSAP): clinical, diagnostic, and epidemiological methodologies. Clin Infect Dis 62(Suppl 1): S9S16.

    • Search Google Scholar
    • Export Citation
  • 13.

    Marks F et al.2017. Incidence of invasive Salmonella disease in sub-Saharan Africa: a multicentre population-based surveillance study. Lancet Glob Health 5: e310e323.

    • Search Google Scholar
    • Export Citation
  • 14.

    Marks F et al., 2021. Pathogens causing acute febrile illness among children and adolescents in Burkina Faso, Madagascar and Sudan. Clin Infect Dis. doi: 10.1093/cid/ciab289.

    • Search Google Scholar
    • Export Citation
  • 15.

    Kodani M , Winchell JM , 2012. Engineered combined-positive-control template for real-time reverse transcription-PCR in multiple-pathogen-detection assays. J Clin Microbiol 50: 10571060.

    • Search Google Scholar
    • Export Citation
  • 16.

    Tabue RN et al.2019. Case definitions of clinical malaria in children from three health districts in the north region of Cameroon. BioMed Res Int 2019: 9709013.

    • Search Google Scholar
    • Export Citation
  • 17.

    Sigurdson AJ , Ha M , Cosentino M , Franklin T , Haque KA , Qi Y , Glaser C , Reid Y , Vaught JB , Bergen AW , 2006. Long-term storage and recovery of buccal cell DNA from treated cards. Cancer Epidemiol Biomarkers Prev 15: 385388.

    • Search Google Scholar
    • Export Citation
  • 18.

    Buckton AJ , Prabhu DP , Cane PA , Pillay D , 2009. No evidence for cross-contamination of dried blood spots excised using an office hole-punch for HIV-1 drug resistance genotyping. J Antimicrob Chemother 63: 615616.

    • Search Google Scholar
    • Export Citation
  • 19.

    Driver GA , Patton JC , Moloi J , Stevens WS , Sherman GG , 2007. Low risk of contamination with automated and manual excision of dried blood spots for HIV DNA PCR testing in the routine laboratory. J Virol Methods 146: 397400.

    • Search Google Scholar
    • Export Citation
  • 20.

    Bonne N , Clark P , Shearer P , Raidal S , 2008. Elimination of false-positive polymerase chain reaction results resulting from hole punch carryover contamination. J Vet Diagn Invest 20: 6063.

    • Search Google Scholar
    • Export Citation
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Detection of Pathogens of Acute Febrile Illness Using Polymerase Chain Reaction from Dried Blood Spots

Brian GrundyDivision of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia;

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Ursula PanznerInternational Vaccine Institute, Seoul, Republic of Korea;
Swiss Tropical and Public Health Institute, Basel, Switzerland;
University of Basel, Basel, Switzerland;

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Jie LiuDivision of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia;

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Hyon Jin JeonInternational Vaccine Institute, Seoul, Republic of Korea;
Cambridge Institute of Therapeutic Immunology and Infectious Disease, University of Cambridge School of Clinical Medicine, Cambridge Biomedical Campus, Cambridge, United Kingdom;

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Justin ImInternational Vaccine Institute, Seoul, Republic of Korea;

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Vera von KalckreuthInternational Vaccine Institute, Seoul, Republic of Korea;

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Frank KoningsInternational Vaccine Institute, Seoul, Republic of Korea;

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Gi Deok PakInternational Vaccine Institute, Seoul, Republic of Korea;

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Ligia Maria Cruz EspinozaInternational Vaccine Institute, Seoul, Republic of Korea;

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Abdramane Soura BassiahiInstitut Supeìrieur des Sciences de la Population, University of Ouagadougou, Burkina Faso;

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Nagla GasmelseedFaculty of Medicine, University of Gezira, Wad Medani, Sudan;

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Raphaël RakotozandrindrainyUniversity of Antananarivo, Antananarivo, Madagascar

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Suzanne StroupDivision of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia;

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Eric R. HouptDivision of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia;

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Florian MarksInternational Vaccine Institute, Seoul, Republic of Korea;
Cambridge Institute of Therapeutic Immunology and Infectious Disease, University of Cambridge School of Clinical Medicine, Cambridge Biomedical Campus, Cambridge, United Kingdom;
University of Antananarivo, Antananarivo, Madagascar

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ABSTRACT.

Quantitative polymerase chain reaction (qPCR) of dried blood spots (DBS) for pathogen detection is a potentially convenient method for infectious disease diagnosis. This study tested 115 DBS samples paired with whole blood specimens of children and adolescent from Burkina Faso, Sudan, and Madagascar by qPCR for a wide range of pathogens, including protozoans, helminths, fungi, bacteria, and viruses. Plasmodium spp. was consistently detected from DBS but yielded a mean cycle threshold (Ct) 5.7 ± 1.6 higher than that from whole blood samples. A DBS qPCR Ct cutoff of 27 yielded 94.1% sensitivity and 95.1% specificity against the whole blood qPCR cutoff of 21 that has been previously suggested for malaria diagnosis. For other pathogens investigated, DBS testing yielded a sensitivity of only 8.5% but a specificity of 98.6% compared with whole blood qPCR. In sum, direct PCR of DBS had reasonable performance for Plasmodium but requires further investigation for the other pathogens assessed in this study.

Acute febrile illness (AFI) is one of the most common reasons for patients to seek healthcare.1 There are many infectious agents, including bacteria, viruses, fungi, and parasites, that require a wide array of diagnostic methods.2 Quantitative polymerase chain reaction (qPCR) provides a sensitive and specific method for pathogen detection.3 However, PCR-based diagnostics are often unavailable in resource-limited settings; therefore, samples must be collected and tested later. Storing and shipping frozen samples can be costly and challenging. Dried blood spots (DBS) are a convenient alternative because only a small volume is required, and storage is typically at ambient temperature. DBS are of use for newborn HIV diagnosis4 and screening for sickle cell disease,5 among many other uses.6 Recent studies have shown the potential of molecular detection of pathogens using DBS including Plasmodium spp.,7,8 dengue fever virus,9 Leptospira spp.,10 Streptococcus spp.11 and Haemophilus spp.11 but with varying success.

The Typhoid Fever Surveillance in Africa Program (TSAP) was a multisite prospective fever surveillance study conducted in 10 sub-Saharan African countries between 2011 and 2013.12 The project’s main goal was to identify Salmonella Typhi and invasive nontyphoidal Salmonella spp. in febrile patients using blood culture-based diagnostics.13 In a recent investigation using whole blood samples from Sudan, Burkina Faso, and Madagascar, we identified by qPCR multiple pathogens not detected previously.14 Here, we compare the performance of qPCR on paired DBS.

A total of 615 whole blood samples from Burkina Faso, Sudan, and Madagascar, were stored at –80°C and tested in 2018 by qPCR by TaqMan Array Card (TAC).14 Of these 615 samples, we selected 107 DBS for which whole blood was positive for one or more of 15 pathogens, in addition to eight DBS for which blood was negative for all pathogens. DBS were prepared on Whatman FTA mini cards (WB120055, Cytiva, Buckinghamshire, UK) from venous blood at the time of enrollment in 2011 and 2013, stored at room temperature at the International Vaccine Institute, South Korea, and tested at the University of Virginia in 2021. The ethical clearance for the underlying TSAP program included additional pathogen identification including this work. The International Vaccine Institute’s Institutional Review Board (IRB), the Comiteì d’Ethique pour la Recherche en Santeì of the Ministry of Health in Burkina Faso, the Comiteì d’Ethique of the Ministry of Health of the Republic of Madagascar, and the National Research Ethics Review Committee of the National Ministry of Health in Sudan provided ethical approvals. The University of Virginia IRB provided additional approval for this work.

DBS were hole punched with a 3-mm Harris Uni-core hole puncher. Hole punchers were cleaned after use by soaking in 70% ethanol and Eliminase (VWR, Avantor, Radnor, PA). Nucleic acid extraction was carried out on six hole punches using the QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. Phocine herpesvirus 1 (PhHV-1; 106 copies) and MS2 bacteriophage (107 copies) were added to monitor extraction and amplification efficiency. Extraction blanks were included to monitor for laboratory contamination.

On the basis of the previous TAC results,14 cognate qPCR assays targeting Aeromonas baumannii, Aeromonas spp., Bartonella spp., cytomegalovirus, dengue virus, enterovirus, Escherichia coli, Histoplasma spp., K. oxytoca, M. tuberculosis, Plasmodium spp., Rickettsia spp., Staphylococcus aureus, Streptococcus pneumoniae, and Schistosoma spp., were performed on 96-well plates as individual reactions. Each assay target was tested on at least 23 samples each, including known positive samples for each target based on blood TAC results and randomly selected negative samples from all 115 extracted DBS. Each 20-µL reaction was carried out with 10 µL nucleic acid extract, 5 µL TaqMan Fast Virus master mix (Life Technologies, Thermo Fisher Scientific, Carlsbad, CA), 900 nM primers, and a 250-nM probe as described in the previous study.14 Cycling conditions (same as TAC) included reverse transcription for 10 minutes at 50°C, denaturation for 20 seconds at 95°C, and 40 PCR cycles of 3 seconds at 95°C and 30 seconds at 60°C. Nuclease-free water and a combined positive control template15 were used as controls.

A sample was considered positive if a reaction yielded a cycle threshold (Ct) of less than 40. DBS Ct-values were compared with whole blood TAC qPCR Ct-values to assess correlation and sensitivity and specificity. Statistical analysis, including frequencies, correlations, and receiver operator curve (ROC) analysis, was carried out using SPSS version 26.

Plasmodium spp. was tested in 95 DBS samples (Figure 1), of which 48 were positive in paired whole blood samples. Of the 48 paired DBS, 44 were positive for Plasmodium spp., yielding a DBS sensitivity of 91.7%. Of the 47 samples that were negative for Plasmodium spp. in whole blood, eight DBS were positive, with higher Cts ranging from 32.5 to 38.9, yielding a specificity of 83.0%. The median Ct for positive DBS samples for Plasmodium spp. was 23.9 (interquartile range [IQR]: 21.9–26.7) compared with a whole blood Ct median of 18.7 (IQR: 16.4–21.7) on TAC.14 The mean increase in DBS Ct for samples positive by whole blood for Plasmodium spp. was 5.7 ± 1.6. A whole blood qPCR Ct-value of 21 on TAC corresponds to a few thousand parasites per microliter, which has been used as a clinical case definition.16 Against this standard, the optimal DBS cutoff was 27 (ROC area under the curve: 0.98) and yielded a sensitivity and specificity of 94.1% and 95.1%, respectively.

Figure 1.
Figure 1.

Correlation of whole blood specimens paired with dried blood spots by qPCR for Plasmodium spp. Line of best fit (y = 1.02x + 5.41, R2 = 0.889) of Ct-values, excluding negative values (Ct = 40). Ct = cycle threshold; DBS = dried blood spot; qPCR = quantitative polymerase chain reaction.

Citation: The American Journal of Tropical Medicine and Hygiene 106, 2; 10.4269/ajtmh.21-0814

Beyond Plasmodium spp., there were 59 whole blood samples that were qPCR positive for one or more of 14 other pathogens. These whole blood Cts were generally high (median: 34.7, IQR: 33.6–36.3). The corresponding DBS were tested with the relevant qPCR assays, and 5/59 (1 E. coli, 2 cytomegalovirus, 1 S. pneumoniae, 1 S. aureus) were positive with Cts ranging from 35.3 to 39.5 leading to 8.5% sensitivity. Specificity of DBS by qPCR was assessed by performing myriad assays (as described in methods) on DBS with paired whole blood negative samples (total PCR reactions = 340), of which four (one cytomegalovirus, two dengue, one E. coli) were DBS PCR-positive with Cts ranging from 34.3 to 39.5, resulting in 98.6% specificity.

This work explored the capabilities of DBS for pathogen detection by qPCR. Although the detection of Plasmodium spp. from DBS correlated reasonably well with detection from whole blood, DBS qPCR was insensitive for the other pathogens of AFI that we evaluated.14 Other studies have shown promising results in Plasmodium spp. detection by DBS PCR and higher Cts compared with whole blood,7,8 but findings have been inconsistent for other pathogens.911 This is likely due to a higher quantity of malaria parasites in blood compared with that of bacteria and certain viruses.

This study was limited by small sample size for many pathogens, and we would also note the long duration of storage of the DBS. Ideally, DBS samples would be tested within a few days to several months.810 Despite this limitation, pathogen DNA was still detected more than 8 years later, especially for Plasmodium spp. Others have also found amplification of DNA from filter paper after 7 years despite storage at room temperature.17 Nonetheless, given the small volume of blood applied onto DBS and the possibility of nucleic acid degradation over time, particularly RNA, studies are needed to evaluate the use of new filter paper products, extraction techniques, and storage conditions. Another observation we noted during pilot testing of purification and amplification methods was that contamination from hole punchers used in DBS analyses is a potential risk,1820 especially for Plasmodium spp. detection. We tested and developed a new cleaning method of hole punchers to minimize the risk of contamination. In sum, direct PCR of DBS has good performance for clinical malaria and could be useful for malaria surveillance but requires further investigation for the other pathogens assessed in this study.

REFERENCES

  • 1.

    Iroh Tam PY , Obaro SK , Storch G , 2016. Challenges in the etiology and diagnosis of acute febrile illness in children in low- and middle-income countries. J Pediatric Infect Dis Soc 5: 190205.

    • Search Google Scholar
    • Export Citation
  • 2.

    Prasad N , Murdoch DR , Reyburn H , Crump JA , 2015. Etiology of severe febrile illness in low- and middle-income countries: a systematic review. PLoS One 10: e0127962.

    • Search Google Scholar
    • Export Citation
  • 3.

    Liu J et al.2016. Development of a TaqMan Array Card for acute-febrile-illness outbreak investigation and surveillance of emerging pathogens, including ebola virus. J Clin Microbiol 54: 4958.

    • Search Google Scholar
    • Export Citation
  • 4.

    Sutcliffe CG , Mutanga JN , Moyo N , Schue JL , Hamahuwa M , Thuma PE , Moss WJ , 2020. Acceptability and feasibility of testing for HIV infection at birth and linkage to care in rural and urban Zambia: a cross-sectional study. BMC Infect Dis 20: 227.

    • Search Google Scholar
    • Export Citation
  • 5.

    Nnodu OE et al.2020. Implementing newborn screening for sickle cell disease as part of immunisation programmes in Nigeria: a feasibility study. Lancet Haematol 7: e534e540.

    • Search Google Scholar
    • Export Citation
  • 6.

    Gupta K , Mahajan R , 2018. Applications and diagnostic potential of dried blood spots. Int J Appl Basic Med Res 8: 12.

  • 7.

    Canier L et al.2015. Malaria PCR detection in Cambodian low-transmission settings: dried blood spots versus venous blood samples. Am J Trop Med Hyg 92: 573577.

    • Search Google Scholar
    • Export Citation
  • 8.

    Zainabadi K , Adams M , Han ZY , Lwin HW , Han KT , Ouattara A , Thura S , Plowe CV , Nyunt MM , 2017. A novel method for extracting nucleic acids from dried blood spots for ultrasensitive detection of low-density Plasmodium falciparum and Plasmodium vivax infections. Malar J 16: 377.

    • Search Google Scholar
    • Export Citation
  • 9.

    Aubry M et al.2012. Use of serum and blood samples on filter paper to improve the surveillance of dengue in Pacific Island countries. J Clin Virol 55: 2329.

    • Search Google Scholar
    • Export Citation
  • 10.

    Musso D , Roche C , Marfel M , Bel M , Nilles EJ , Cao-Lormeau VM , 2014. Improvement of leptospirosis surveillance in remote Pacific Islands using serum spotted on filter paper. Int J Infect Dis 20: 7476.

    • Search Google Scholar
    • Export Citation
  • 11.

    Selva L et al.2013. Detection of Streptococcus pneumoniae and Haemophilus influenzae type B by real-time PCR from dried blood spot samples among children with pneumonia: a useful approach for developing countries. PLoS One 8: e76970.

    • Search Google Scholar
    • Export Citation
  • 12.

    von Kalckreuth V et al.2016. The Typhoid Fever Surveillance in Africa Program (TSAP): clinical, diagnostic, and epidemiological methodologies. Clin Infect Dis 62(Suppl 1): S9S16.

    • Search Google Scholar
    • Export Citation
  • 13.

    Marks F et al.2017. Incidence of invasive Salmonella disease in sub-Saharan Africa: a multicentre population-based surveillance study. Lancet Glob Health 5: e310e323.

    • Search Google Scholar
    • Export Citation
  • 14.

    Marks F et al., 2021. Pathogens causing acute febrile illness among children and adolescents in Burkina Faso, Madagascar and Sudan. Clin Infect Dis. doi: 10.1093/cid/ciab289.

    • Search Google Scholar
    • Export Citation
  • 15.

    Kodani M , Winchell JM , 2012. Engineered combined-positive-control template for real-time reverse transcription-PCR in multiple-pathogen-detection assays. J Clin Microbiol 50: 10571060.

    • Search Google Scholar
    • Export Citation
  • 16.

    Tabue RN et al.2019. Case definitions of clinical malaria in children from three health districts in the north region of Cameroon. BioMed Res Int 2019: 9709013.

    • Search Google Scholar
    • Export Citation
  • 17.

    Sigurdson AJ , Ha M , Cosentino M , Franklin T , Haque KA , Qi Y , Glaser C , Reid Y , Vaught JB , Bergen AW , 2006. Long-term storage and recovery of buccal cell DNA from treated cards. Cancer Epidemiol Biomarkers Prev 15: 385388.

    • Search Google Scholar
    • Export Citation
  • 18.

    Buckton AJ , Prabhu DP , Cane PA , Pillay D , 2009. No evidence for cross-contamination of dried blood spots excised using an office hole-punch for HIV-1 drug resistance genotyping. J Antimicrob Chemother 63: 615616.

    • Search Google Scholar
    • Export Citation
  • 19.

    Driver GA , Patton JC , Moloi J , Stevens WS , Sherman GG , 2007. Low risk of contamination with automated and manual excision of dried blood spots for HIV DNA PCR testing in the routine laboratory. J Virol Methods 146: 397400.

    • Search Google Scholar
    • Export Citation
  • 20.

    Bonne N , Clark P , Shearer P , Raidal S , 2008. Elimination of false-positive polymerase chain reaction results resulting from hole punch carryover contamination. J Vet Diagn Invest 20: 6063.

    • Search Google Scholar
    • Export Citation

Author Notes

Address correspondence to Brian Grundy, Division of Infectious Diseases and International Health, University of Virginia, 345 Crispell Drive, Charlottesville, VA 22908. E-mail: bsg3md@hscmail.mcc.virginia.edu

Financial support: This work was supported by the National Institutes of Health (NIH; K24AI102972 to E. H.). B. G. is supported by the NIH (T32 AI007046). The Bill & Melinda Gates Foundation provided financial support for the TSAP study (OPP1127988).

Authors’ addresses: Brian Grundy, Jie Liu, Suzanne Stroup, and Eric Houpt, Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA, E-mails: bsg3md@hscmail.mcc.virginia.edu, jl5yj@virginia.edu, ses8d@virginia.edu, and erh6k@hscmail.mcc.virginia.edu. Ursula Panzner, Justin Im, Frank Konings, Vera von Kalckreuth, Gi Deok Pak, Ligia Maria Cruz Espinoza, and Florian Marks, International Vaccine Institute, Seoul, Republic of Korea, E-mails: upanzner@ivi.int, justin.im@ivi.int, fkonings@gmail.com, vera.vkalckreuth@web.de, gdpak@ivi.int, lcruz@ivi.int, and fmarks@ivi.int. Hyon Jin Jeon, Cambridge Institute of Therapeutic Immunology and Infectious Disease, University of Cambridge School of Clinical Medicine, Cambridge Biomedical Campus, Cambridge, UK, E-mail: hjj32@cam.ac.uk. Abdramane Soura Bassiahi, Institut Supérieur des Sciences de la Population, Ouagadougou, Burkina Faso, E-mail: bassiahi@gmail.com. Nagla Gaslmelseed, Faculty of Medicine, University of Gezira, Wad Medani, Sudan, E-mail: nag_la@yahoo.com. Raphaë¨l Rakotozandrindrainy, University Antananarivo, Antananarivo, Madagascar, E-mail: rakrapha13@gmail.com.

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