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Volume 25, Number 5—May 2019

Management of Central Nervous System Infections, Vientiane, Laos, 2003–2011

Audrey Dubot-PérèsComments to Author , Mayfong Mayxay, Rattanaphone Phetsouvanh1, Sue J. Lee, Sayaphet Rattanavong, Manivanh Vongsouvath, Viengmon Davong, Vilada Chansamouth, Koukeo Phommasone, Catrin Moore, Sabine Dittrich, Olay Lattana, Joy Sirisouk, Phonelavanh Phoumin, Phonepasith Panyanivong, Amphonesavanh Sengduangphachanh, Bountoy Sibounheuang, Anisone Chanthongthip, Manivone Simmalavong, Davanh Sengdatka, Amphaivanh Seubsanith, Valy Keoluangkot, Prasith Phimmasone, Kongkham Sisout, Khamsai Detleuxay, Khonesavanh Luangxay, Inpanh Phouangsouvanh, Scott B. Craig, Suhella M. Tulsiani, Mary-Anne Burns, David A.B. Dance, Stuart D. Blacksell, Xavier de Lamballerie, and Paul N. Newton
Author affiliations: Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit, Mahosot Hospital, Vientiane, Laos (A. Dubot-Pérès, M. Mayxay, R. Phetsouvanh, S. Rattanavong, M. Vongsouvath, V. Davong, V. Chansamouth, K. Phommasone, C. Moore, S. Dittrich, O. Lattana, J. Sirisouk, P. Phoumin, P. Panyanivong, A. Sengduangphachanh, B. Sibounheuang, A. Chanthongthip, M. Simmalavong, D. Sengdatka, A. Seubsanith, D.A.B. Dance, P.N. Newton); University of Oxford Nuffield Department of Clinical Medicine Center for Tropical Medicine and Global Health, Oxford, UK (A. Dubot-Pérès, S.J. Lee, C. Moore, S. Dittrich, D.A.B. Dance, S.D. Blacksell, P.N. Newton); Unité des Virus Émergents (UVE: Aix-Marseille Univ-IRD 190-INSERM 1207-IHU Méditerranée Infection), Marseille, France (A. Dubot-Pérès, X. de Lamballerie); University of Health Sciences Institute of Research and Education Development, Vientiane (M. Mayxay); Mahidol University Faculty of Tropical Medicine Mahidol– Oxford Tropical Medicine Research Unit, Bangkok, Thailand (S.J. Lee, S.D. Blacksell); Mahosot Hospital, Vientiane (V. Keoluangkot, P. Phimmasone, K. Sisout, K. Detleuxay, K. Luangxay, I. Phouangsouvanh); Queensland Health Forensic and Scientific Service World Health Organization Collaborating Centre for Reference and Research on Leptospirosis, Brisbane, Queensland, Australia (S.B. Craig, S.M. Tulsiani, M.-A. Burns); London School of Hygiene and Tropical Medicine Faculty of Infectious and Tropical Diseases, London, UK (D.A.B. Dance, P.N. Newton)

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Table 2

Diagnostic laboratory tests used to confirm etiology in patients with suspected central nervous system infection, by sample type, Laos, January 2003–August 2011*

Pathogens tested Cerebrospinal fluid† Blood
Malaria pathogens
Giemsa thick and thin smear (if patient from endemic area)
Leptospira spp.
Hydrolysis probe real-time PCR (19)
Culturing of blood clot on EMJH medium; microscopic agglutination test of admission and follow-up serum samples (4-fold rise considered positive result) (20); hydrolysis probe real-time RT-PCR from buffy coat (19)
Cryptococcus spp.
Indian ink stain; if HIV suspected, Cryptococcus Antigen Latex Agglutination Test (IMMY,; if Indian ink positive and HIV suspected, culture on Sabouraud agar
Mycobacterium tuberculosis
Lowenstein-Jensen culture; auramine and Ziehl-Neelsen stains
Rickettsia spp.‡
Hydrolysis probe real-time PCR (21,22)
Hydrolysis probe real-time and conventional PCR from buffy coat (21,22); sequencing
R. typhi, Orientia tsutsugamushi
Hydrolysis probe real-time PCR (21,23)
Hydrolysis probe real-time PCR on buffy coat (21,23); IgM and IgG indirect immunofluorescent assay of admission and follow-up serum samples (4-fold rise considered positive result) (24)
Community-acquired bacterial infection
Gram stain; culture in blood agar and chocolate agar
Blood culture bottle
Streptococcus pneumoniae, Streptococcus suis, Haemophilus influenzae, Neisseria meningitidis
Culture on blood agar, chocolate agar, and MacConkey plates (for patients <1 year of age); 
hydrolysis probe real-time RT-PCR (2527)
Blood culture bottle
Dengue virus
Hydrolysis probe real-time RT-PCR (28); NS1 ELISA (Dengue Early ELISA, Panbio,; ELISA IgM (Japanese Encephalitis/Dengue IgM Combo ELISA, Panbio)
Hydrolysis probe real-time RT-PCR on serum samples (28); NS1 ELISA on serum samples; ELISA IgM on admission and follow-up serum samples (assessed seroconversion)
ELISA IgM (Japanese Encephalitis/Dengue IgM Combo ELISA, Panbio)
ELISA IgM on admission and follow-up serum samples (assessed seroconversion)
Enterovirus, West Nile virus, influenza viruses A and B, Henipavirus
Hydrolysis probe real-time RT-PCR (2931) (in house)
Hydrolysis probe real-time RT-PCR on serum samples (2931) (in house)
SYBR Green real-time RT-PCR (32,33)
SYBR Green real-time RT-PCR on serum samples (32,33)
Herpes simplex virus 1 and 2, Varicella zoster virus, human cytomegalovirus
Hydrolysis probe real-time RT-PCR (3436)
Measles virus, mumps virus
Hydrolysis probe real-time RT-PCR (37,38); if seropositive in blood sample, IgM ELISA with Enzygnost Anti-Measles Virus/IgM or Enzygnost Anti-Parotidis/IgM kits (Dade Behring,
Hydrolysis probe real-time RT-PCR on serum samples (37,38); IgM and IgG ELISAs: Enzygnost Anti-Measles Virus/IgM, Enzygnost Anti-Measles Virus/IgG, Enzygnost Anti-Parotidis/IgM, and Enzygnost Anti-Parotidis/IgG kits (Dade Behring) (assessed seroconversion)
HIV None Determine HIV-1/2 Combo (Alere, or Uni-Gold HIV (Trinity Biotech,

*See Appendix ( for further details on methods. Cerebrospinal fluid and serum samples were inoculated on Vero and buffalo green monkey kidney cells for viral isolation. The median (interquartile range) interval between admission and follow-up serum sample collection was 9 (6–16) days. EMJH, Ellinghausen-McCullough-Johnson-Harris; JEV, Japanese encephalitis virus; NS1, nonstructural protein 1; RT-PCR, reverse transcription PCR.
†Contraindications for lumbar puncture included focal neurologic signs on examination, clinical evidence for raised intracranial pressure, skin or soft tissue sepsis at the proposed puncture site, severe coagulopathy, or severe thrombocytopenia.
‡Buffy coats were inoculated on Vero and L929 cells for O. tsutsugamushi and Rickettsia sp isolation.
§Detection of JEV IgM in a single sample of serum is considered as laboratory confirmation according to World Health Organization criteria. However, in this study, to be conservative and consistent with interpretation of other test results, a single detection of JEV IgM in serum was not counted as confirming JEV central nervous system infection.
¶Flavivirus RT-PCR would have likely detected the main flaviviruses circulating in Laos.

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