Causes of non-malarial fever in Laos: a prospective study

Summary Background Because of reductions in the incidence of Plasmodium falciparum malaria in Laos, identification of the causes of fever in people without malaria, and discussion of the best empirical treatment options, are urgently needed. We aimed to identify the causes of non-malarial acute fever in patients in rural Laos. Methods For this prospective study, we recruited 1938 febrile patients, between May, 2008, and December, 2010, at Luang Namtha provincial hospital in northwest Laos (n=1390), and between September, 2008, and December, 2010, at Salavan provincial hospital in southern Laos (n=548). Eligible participants were aged 5–49 years with fever (≥38°C) lasting 8 days or less and were eligible for malaria testing by national guidelines. Findings With conservative definitions of cause, we assigned 799 (41%) patients a diagnosis. With exclusion of influenza, the top five diagnoses when only one aetiological agent per patient was identified were dengue (156 [8%] of 1927 patients), scrub typhus (122 [7%] of 1871), Japanese encephalitis virus (112 [6%] of 1924), leptospirosis (109 [6%] of 1934), and bacteraemia (43 [2%] of 1938). 115 (32%) of 358 patients at Luang Namtha hospital tested influenza PCR-positive between June and December, 2010, of which influenza B was the most frequently detected strain (n=121 [87%]). Disease frequency differed significantly between the two sites: Japanese encephalitis virus infection (p=0·04), typhoid (p=0·006), and leptospirosis (p=0·001) were more common at Luang Namtha, whereas dengue and malaria were more common at Salavan (all p<0·0001). With use of evidence from southeast Asia when possible, we estimated that azithromycin, doxycycline, ceftriaxone, and ofloxacin would have had significant efficacy for 258 (13%), 240 (12%), 154 (8%), and 41 (2%) of patients, respectively. Interpretation Our findings suggest that a wide range of treatable or preventable pathogens are implicated in non-malarial febrile illness in Laos. Empirical treatment with doxycycline for patients with undifferentiated fever and negative rapid diagnostic tests for malaria and dengue could be an appropriate strategy for rural health workers in Laos. Funding Wellcome Trust, WHO–Western Pacific Region, Foundation for Innovative New Diagnostics, US Centers for Disease Control and Prevention.

Microscopy Samples were packed in plastic screw capped tubes with paper wadding, in double skinned locked metal boxes (Blacksell et al. 2006). LNT specimens were transported at ambient temperature by flight (~90 minutes) or by bus (~24 h) to Vientiane and SV specimens by bus (~24 h). Temperature loggers (TinyTag Transit-2 TG0050, Gemini Data Loggers (UK)) were kept in each metal box from October 2009, to record temperature every 2 hours. The mean (+/-2SD; range) temperatures measured within the transport boxes (for 15 months) were 25 (15.6-34.4; 11.0-44.2) o C for ) o C for SV (T test, P<0.0001).

B. Malaria slides and RDTs
Giemsa-stained malaria smears and pLDH-based ICT Malaria Combo Cassette Test (ICT Diagnostics, Cape Town, South Africa) were performed for all patients. Slides were re-checked by a Centre for Malariology, Parasitology & Entomology (CMPE) World Health Organization (WHO)-accredited Level 1 microscopist, blinded to the results from the provincial hospitals.

C. Blood cultures
Blood culture bottles (Pharmaceutical Factory No. 2, Vientiane) 3 were weighed to estimate the volume of blood added to the bottles (specific gravity of blood assumed=1.06 (Trudnowski & Rico 1974)). Bottles from each batch were inoculated with reference organisms (Pseudomonas aeruginosa, S. pneumoniae and H. influenzae) to check for their growth and without inoculation, for no growth. The Mahosot laboratory participates in the UK NEQAS General Bacteriology and Antimicrobial Susceptibility Testing scheme. Positive cultures were identified using conventional techniques 3 and antibiotic susceptibility determined by disc diffusion using Clinical and Laboratory Standards Institute (CLSI) criteria. 14 True bacteraemia and blood culture contaminants were defined using conventional criteria.3 Blood culture results were reported as soon as any clinically relevant information on bacterial growth was available and may therefore have influenced patient management.

D. Dengue/JEV ELISAs
The following Panbio Inc. (now Inverness Inc.) ELISA kits were used to investigate dengue and JEV infection (Jacobson et al. 2007, Moore et al. 2012 The procedure followed that of Phetsouvanh et al. (2009). 17 Dried blood spots were collected onto Proteinsaver filter paper strips and transported and stored in sealed plastic bags, containing silica gel crystals. In Vientiane a cardpunch was used to cut 6 mm diameter discs from the blood-impregnated filter paper blood spots, halfway between the center and the edge of the blood spot. These were eluted overnight in 250l autoclaved PBS at 37C. Saturated discs were equivalent to a 1/25 dilution of serum. Measurements with a NanoDrop spectrophotometer (Thermo Scientific) were done after each extraction to assess the concentration and the purity of the extracted DNA. The extracts were then divided in 2 aliquots (for subsequent PCR duplicates) and kept at -80°C.

RNA
The first

O. Leptospiral Culture
Leptospire culture was performed using the clot remaining after centrifugation of the blood sample, collected in the tube containing no anticoagulant tube, after serum was removed, as described by Wuthiekanun et al. (2007). 16 It is important to note that such clot is not the optimal sample for leptospiral culture 16 but it was the only sample type available. This work was started in August 2009 and therefore the data presented here represent 16 months of leptospiral culture.
Culture of leptospires from clot was performed using 3 ml of Ellinghausen, McCullough, Johnson and Harris (EMJH) medium supplemented with 3% rabbit serum and 0.1% agarose in 5-ml sterile plastic flat-based screw-cap tubes (Sterilin, Barloworld Scientific Ltd., United Kingdom). Three milliliters of EMJH was added to the blood clot remaining after centrifugation of ~ 5 ml whole blood, and removal of serum, using a sterile pipette and left overnight at room temperature. The next morning, the supernatant was transferred into a new 5-ml tube and incubated at Lao room temperature (~ 25 0 C) for 12 weeks. Leptospires were identified by dark-field microscopy at x200

S. Concordance
For the PCR assays the concordance between duplicate runs was high (Supplementary Material 3).

W. Specimen exchange
In order to check the comparability of malaria PCR results, samples were exchanged between Institute Pasteur, Cambodia (IPC) and LOMWRU. Of 16 DNA extracts of red blood cell pellets sent to IPC for malaria PCR, there was 100% concordance between the IPC and LOMWRU results.
IPC used a nested PCR approach targeting the cytb gene followed by sequencing (Steenkeste et al. 2009). Of 18 RNA extracts from sera sent to IP from LOMWRU, 17/18 (94%) concordance was found with one serum, dengue PCR positive in LOMWRU, negative in IPC. IPC used a multiplex nested reverse transcriptase PCR for dengue diagnosis (Lanciotti et al. 1992). Importantly, as we did not send primary specimen types these sample exchanges do not examine differences in nucleic acid extraction.

Supplementary Material -2 Algorithm to determine each patient's infection status according to the results from JE/Dengue IgM Combo ELISA, dengue IgG capture ELISA (HL-IgG), dengue IgG indirect ELISA (LL-IgG) and dengue Early ELISA (detects NS1).
The table below was used to interpret the results of the 3 ELISA kits JEV/Dengue IgM Combo, dengue IgG capture and dengue Early for distinguishing dengue, JEV and flavivirus infections.
For patients with anti-dengue IgM or HL-IgG positive on admission but who were negative in both kits using convalescent sera (when taken within 30 days of admission serum), after repetition, we concluded that they did not have dengue infection and assumed that the admission results represented a nonspecific immune response.
A FALSE result can be a low positive. In case of FALSE IgM or HL-IgG result in admission with negative or still FALSE result in convalescent sample, the infection status is "negative" (a low positive in admission should have been positive in convalescent sample). If IgM or HL-IgG are negative in admission sample then become FALSE in convalescent or in the case they are FALSE in admission with no convalescent sample the infection status stay "uncertain".
Primary and secondary dengue were distinguished according to the following: Primary dengue: LL-IgG negative in acute sample.
In other cases, like IgM positive and IgG positive in admission, because we are not sure that is an acute dengue, it could be a primary or a secondary, we reported as "unknown" for primary or secondary.

Supplementary Material -10. Serology and PCR assays for dengue and JEV and influenza PCR.
Influenza diagnosis was only conducted on samples collected from LNT for 6 months.