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

Background Because of reductions in the incidence of Plasmodium falciparum malaria in Laos, identifi cation 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 defi nitions of cause, we assigned 799 (41%) patients a diagnosis. With exclusion of infl uenza, the top fi ve diagnoses when only one aetiological agent per patient was identifi ed 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 infl uenza PCR-positive between June and December, 2010, of which infl uenza B was the most frequently detected strain (n=121 [87%]). Disease frequency diff ered signifi cantly 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 ofl oxacin would have had signifi cant effi cacy for 258 (13%), 240 (12%), 154 (8%), and 41 (2%) of patients, respectively. Interpretation Our fi ndings suggest that a wide range of treatable or preventable pathogens are implicated in nonmalarial febrile illness in Laos. Empirical treatment with doxycycline for patients with undiff erentiated 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 Pacifi c Region, Foundation for Innovative New Diagnostics, US Centers for Disease Control and Prevention

Development of an improved RT-qPCR Assay for detection of <i>Japanese encephalitis virus</i> (JEV) RNA including a systematic review and comprehensive comparison with published methods - Fig 1

<p>Standard curves of the 1) Pyke, 2) NS2A assays and 3) NS3 assays with G3-RP9-190 on (A) Day 1 and repeated on (B) Day 2. Result of the RT-qPCR run, ‘Cq’, is plotted against the ‘log starting copies number’, at the RNA dilutions detected: Pyke assay 1:10 serial dilutions of G1-769 in triplicate at 10⁻³ to 10⁻⁶; NS2A assay 1:10 serial dilutions of G1-769 in triplicate at 10⁻³ to 10⁻⁷; and NS3 with G3-RP-190 10⁻⁴ to 10⁻⁷. Efficiency = 10^−1/slope-1. R² = Correlation Coefficient. RT-qPCR performed with Fastvirus kit (TaqMan® Fast Virus 1-Step) with a reaction volume of 50μL, sample volume of 30μl, and primer and probe concentrations of 600nM and 300nM respectively. Thermocycling conditions were 50°C for 5 minutes, 95°C for 20 seconds and 45 x (95°C for 15 seconds + x°C for 60 seconds). The optimal annealing temperature ‘x°C’ was different for each assay: 62°C, 60°C and 56°C for the Pyke, NS2A and NS3 assays respectively.</p

Optimisation experiments performed for the three best performing RT-qPCR systems^*.

<p>Optimisation experiments performed for the three best performing RT-qPCR systems^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194412#t003fn001" target="_blank">*</a>}.</p

Cq results for validation of the optimised JEV RT-qPCR assays.

<p>Cq results for validation of the optimised JEV RT-qPCR assays.</p

Standard reaction mixes and cycling conditions.

<p>Standard reaction mixes and cycling conditions.</p

Evaluation of the three optimised RT-qPCR assays using patient samples with Central Nervous System (CNS) infections.

<p>Patients with RT-qPCR ‘positive’ test results.</p

Primers and probes evaluated during the study.

<p>Primers and probes evaluated during the study.</p

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

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