Methods
Investigation Sites and Procedures
MSF has been working in Myanmar's Rakhine State since 1993. Primary health care (PHC), including the diagnosis and treatment of malaria, is provided free of charge at two clinics, in Sittwe and Thetkalpyin, located in the eastern region of Rakhine. All patients presenting at the Sittwe and Thetkalpyin clinics with fever or a history of fever in the prior 24 hours were immediately tested for malaria on capillary blood with the SD 05FK60 RDT. In addition, a slide was prepared from the same capillary sample and examined separately in the laboratory regardless of the SD 05FK60 result. The laboratory technician was not aware of the SD 05FK60 result when examining the smear. Pregnant women screened for malaria as part of antenatal care received the same procedure but were not included in this analysis.
This routine parallel testing of the described population was carried out from 25 October, 2010 until 23 December, 2011 with one month of data lost (23 February to 28 March 2011) as a result of security incidents in eastern Rakhine State.
Microscopy
Thick and thin blood films were prepared and stained with 10% Giemsa solution (pH 7.2) for 10 min and examined by light microscopy using x1,000 magnification. At least 200 fields had to be examined before designating a sample 'negative', or more accurately, 'no malaria parasites seen'. Positive findings were graded on the thick smear using the 'plus' system scale: + (1 to 9 trophozoites in 100 fields); ++ (1 to 10 trophozoites in 10 fields); +++ (1 to 10 trophozoites per field); ++++ (>10 trophozoites per field). These scores were used to estimate parasite densities: + = 10 to 90 parasites/μl; ++ = 100 to 1,000 parasites/μl, +++ = 1,000 to 10,000 parasites/μl; ++++ = >10,000 parasites/μl, assuming a white blood cell count of 8,000/μl. The species was identified using the thin smear.
Rapid Diagnostic Test
The SD 05FK60 RDT is a three-band lateral-flow immunochromatographic antigen detection test in a cassette format and testing was carried out according to the manufacturer's instructions. Readings were taken in daylight, assisted by standard electric lighting. A negative result is indicated by the presence of a single line, the 'C' control line, in the result window. A P. falciparum-positive result was indicated by presence of two colour bands, the P. falciparum test line and the 'C' control line. The presence of the 'Pan' test line and the 'C' control line (again, two colour bands) indicated a P. vivax, P. ovale. or P. malariae-positive result or a mixed non-falciparum infection. The presence of three colour bands, the 'P. falciparum' and 'Pan' test lines and the 'C' control line indicates a P. falciparum-positive result or a mixed infection. In cases where the control line did not appear, the results were interpreted as invalid and the test repeated with a new device.
Statistical Analysis
Data were entered in an Excel file and analysed using Stata 11.0 statistical software (Stata Corporation, College Station, Texas, USA). Samples with pure gametocytaemia were considered negative. Microscopically identified mixed infections were considered separately (Table 1) and not included in the calculations of test accuracy (Table 2). Sensitivity, specificity and predictive values were calculated with 95% confidence intervals (CI) for the detection of P. falciparum and non-falciparum species separately. When calculating the performance parameters for P. falciparum all negative samples plus all P. falciparum positive samples by microscopy contributed to the analysis. Similarly for the non-falciparum analysis all negative samples and all non-falciparum positive samples by microscopy contributed to the analysis. Likelihood ratios were calculated using the following formulae: LR + = sensitivity/(1–specificity) and LR– = (1–specificity)/sensitivity (Table 2).
Quality Control
All malaria RDTs purchased by MSF must pass the WHO lot-testing programme before being shipped to field sites. In addition, the programme follows the WHO Malaria Microscopy Quality Assurance recommendation. In accordance with this protocol, slides were sent to the Shoklo Malaria Research Unit (SMRU) in Mae Sot, Thailand, for external quality control.
Ethical Statement
This manuscript reports a descriptive retrospective analysis of routinely collected data, and therefore no protocol was submitted for ethical review. Data collected contained no personal identifiers.