Background: Malaria is one of the world killers, but it is curable disease if patients have access to early diagnosis and prompt treatment. The aims of this study are to estimate the frequency of malaria cases among febrile patients, to evaluate the frequency of the parasite species, and to detect the best method for diagnosis of malaria in Red Sea State, Sudan. Materials and Methods: This study was carried out in laboratory of the Red Sea Medical center, Port Sudan, Sudan in period from July 2005 to May 2011. Blood samples were collected from 9,670 febrile patients suspected to have malaria and were examined by expert technologists under the supervision of senior pathologist. Another 717 blood samples seen in peripheral laboratories by routine microscopy were sent for confirmation by expert microscopy. Rapid diagnostic tests (RDT) were also done to these blood samples. Results: Out of the 9,670 febrile patients, only 283 had malaria by expert microscopy. The commonest plasmodium specie that causes malaria in the region was P. falciparum (50.2%), followed by P. vivax (43.8%). The specificity of routine microscopy was (52.7%) and that for RDT was (90.4%). The sensitivity of routine microscopy was (98.6%) while that for RDT was (99.8%). Conclusions: Small number of patients complaining of fever had malaria in Red Sea State with P. falciparum representing (50.2%), followed by P. vivax (43.8%). RDT is recommended for diagnosis of malaria in our region.
Keywords: Malaria, P. falciparum, P. vivax, Port Sudan, rapid tests
Malaria incidence in Sudan was estimated to be about 9 million episodes in 2002, and the number of deaths due to malaria was about 44,000.  One of the major contributing factors to malaria mortality is delayed or inaccurate diagnosis.  That is why one of the main strategic directions of the Roll Back Malaria strategic plan for Sudan, early diagnosis and treatment of malaria, is a necessary component in the control of malaria.  Early diagnosis has become even more important after the emergence of drug resistance.  Clinical diagnosis of malaria is imprecise but remains the basis of therapeutic care for the majority of febrile patients in malaria-endemic areas, where laboratory support is often out of reach. Scientific quantification or interpretation of the effects of malaria misdiagnosis on treatment decision, epidemiologic records, or clinical studies has not been adequately investigated.  Rational therapy of malaria is essential to avoid non-target effects, to delay the advent of resistance, and to save cost on alternative drugs. Accurate diagnosis is the only way of effecting rational therapy. Confirmatory diagnosis before treatment initiation recently regained attention, partly influenced by the spread of drug resistance and thus the requirement of more expensive drugs unaffordable to resource-poor countries. 
To our knowledge, this is the first documented work, which evaluates the frequency, the species and the sensitivity of the diagnostic methods of malaria in Red Sea State, Sudan.
Two diagnostic methods are widely used in the detection of the malaria parasite. These are the Giemsa microscopy and rapid diagnostic tests; each has its characteristic strengths and limitations. Until recently, the diagnosis of malaria in clinical laboratories in the Red Sea State has depended almost exclusively on microscopy and this technique remains the most widely used. No previous evaluation for the RDT was done in our region.
The aims of this study are to estimate the frequency of malaria cases among febrile patients, to evaluate the frequency of the parasite species, and to detect the best method for diagnosis of malaria in Red Sea State, Sudan.
This is a prospective study, which was carried in the laboratory of the Red Sea Medical center (RSMC), Port Sudan, Sudan in the period from July 2005 to May 2011. In this laboratory, all blood samples were examined by expert technologists under the supervision of senior pathologist.
Two groups of patients were examined in this research. The blood samples of the first group were collected from febrile patients in RSMC, and they were used to estimate the frequency of malaria infection and the parasite species. In the second group, the blood samples were referred from peripheral laboratories for assessment of the accuracy of the diagnostic methods (routine microscopy and RDT) used in the region.
In the first group, blood samples were collected directly from 9670 febrile patients suspected to have malaria, in the RSMC laboratory. These blood samples were then examined by expert microscopist. In positive blood films the specie, the stages, and the degree of parasitemia were identified.
In the second group, 717 blood samples seen in peripheral laboratories – from 3 areas(Area 1, 2, and 3) by routine microscopy and were sent for confirmation by expert microscopy to the Red Sea Medical center laboratory. Rapid diagnostic tests (RDT) were also done to these blood samples.
An informed consent was taken from all patients participating in this study. Ethical clearance was approved from the local Ethical Review committee (ERC).
For routine microscopy: Thick and thin blood smears were prepared for each patient’s blood sample in this study. The conventional methods of Giemsa staining were used for the prepared slides.
For the RDT [SD malaria P.f/P.v, www.standardia.com, Korea], blood specimen is applied to the sample pad on the test card along with 3-4 drops of assay diluent. After 5-20 minutes, the presence of specific bands in the test card window indicate whether the patient is infected with Plasmodium falciparum or one of the other 3 species of human malaria. A control band should appear in all RDT.
Expert microscopic examination of Giemsa-stained blood film is currently the standard method for malaria diagnosis.  The criteria for expert microscopy were as follow: Thick and thin blood films were stained with 10% Giemsa stain for 10 min and examined by two experienced microscopists who had no knowledge of patients’ disease status (blind examination) to avoid any bias in blood film readings. The microscopist counted a minimum of 100 consecutive fields in the thick blood film before classifying a slide as negative.
Statistical analysis : Data were analyzed by using a computer Statistical Package for Social Sciences (SPSS) program version 16. Results are presented as frequency and percentage.
In this study, two groups of patients were examined. The first group constitutes 9,670 patients. Patients included here were between 2 and 50 years old and were complaining of fever (≥ 38°C) or gave history of fever (within 48 hours). The ratio of male to female patients was 0.9:1. All patients who had been treated for malaria in the previous 4 weeks were excluded from the study.
Out of the 9,670 febrile patients, 283 had malaria by expert microscopy. [Table 1] show that the commonest plasmodium specie that caused malaria in the region was P. falciparum (50.2%), followed by P. vivax (43.8%).
The study of the diagnostic accuracy of the methods used for detection of the malaria parasite was illustrated by flow diagram in [Figure 1]. Out of the 717 blood samples referred from the peripheral areas, 108 samples were positive by routine microscopy (34 samples were referred from peripheral area 1, 27 samples were referred from peripheral area 2, and 47 samples were referred from peripheral area 3). The confirmed positive malaria cases by expert microscopy were 57 samples. Therefore, the specificity of routine microscopy in the peripheral areas of Red Sea State was 52.7%. 609 samples were reported as negative by the routine microscopy. The confirmed negative samples were 601 (98.6% sensitivity).
The overall positive RDT (from all of the 3 peripheral areas) were 70 samples, compared to 64 samples confirmed positive by expert microscopy in these samples. Therefore, the specificity of the RDT was found to be 90.4%. 647 samples read negative result by RDT. The confirmed negative samples were 646 (99.8% sensitivity) with a single patient read false negative.
Red sea State is an endemic area for multiple febrile illnesses. Dengue fever, respiratory tract infections, gastroenteritis, and urinary tract infections represent diseases that commonly cause fever in the region.  Therefore, it is not strange to find only 283 positive malaria cases among the 9670 febrile patients. The interesting observation is that, most of these positive malaria patients gave history of travelling to outside Port Sudan (the capital of Red Sea State) at least 2 weeks before the fever appear. These patients had the possibility of contracting the disease from outside the study area. The reason for this low frequency of malaria cases may be related to the amount of anopheles mosquito in the region. Unfortunately, no data regarding the occurrence and the breading sites of anopheles mosquito were available.
Plasmodium falciparum was the commonest specie that caused malaria in Red Sea State (50.2%), followed by P. vivax (43.8%). P. falciparum is more prevalent in sub-Saharan Africa than in other regions of the world; in most African countries, more than 75% of cases were due to P. falciparum, whereas in most other countries with malaria transmission, other less virulent Plasmodial species predominate.  P. vivax is found mainly in the United States, Latin America, and in some parts of Africa. Overall, it accounts for 65% of malaria cases in Asia and South America. , According to our finding, high frequency (43.8%) of P. vivax was present in the studied area compared to other African regions.
Giemsa microscopy is regarded as the most suitable diagnostic instrument for malaria control because it is inexpensive to perform, able to differentiate malaria species, and quantify parasites. However, microscopy requires well-trained, competent microscopists and rigorous maintenance of functional infrastructures plus effective quality control (QC) and quality assurance (QA).  In comparison to expert microscopy, poor specificity (52.7%) of routine microscopy in the peripheral areas was found in this study. Poor blood film preparation generates artifacts, commonly mistaken for malaria parasites, including bacteria, fungi, stain precipitation, dirt, and cell debris. 
In this study, the specificity of the RDT was 90.4%. There were 6 false-positive RDT results. Occasional false-positive results due to the presence of rheumatoid factor have previously been reported with diagnostic devices based on the detection of HRP II.  Furthermore, detection of antigen may persist for up to 28 days after cure of infection.  The test devices, which were used, contain plasmodium antigens that could detect P. falciparum and P. vivax (plus the other species). Test devices of RDT that could detect only P. falciparum will give high false negative results in the patient infected with P. vivax (43.8%) and hence should not be used in Red Sea State.
The sensitivity of the RDT in our region was 99.8%. Because of the high specificity and sensitivity, RDT is recommended to be the test of choice for diagnosis of malaria in the region. Positive RDT may be followed by microscopy examination for two reasons. First, for detection of the parasite specie in the infected patient. The currently approved RDT detects 2 different malaria antigens; one is specific for P. falciparum and the other is found in all 4 human species of malaria. Thus, microscopy is needed to determine the species of malaria that was detected by the RDT. In addition, microscopy is needed to quantify the proportion of red blood cells that are infected, which is an important prognostic indicator.
The cost-effectiveness of this strategy vary with RDT cost, malaria blood film cost, cost of anti-malarial treatment, and the cost of treatment of other febrile illnesses when malaria has been ruled out. This strategy become more cost-effective as the price of anti-malarials goes up.
To provide accurate diagnosis of malaria, two important issues should be available in the region. First, an effective Quality Control (QC)/Quality Assurance (CA) system engaging different organizational levels is needed. This involves standardization of procedures and establishment of national-level diagnostics centers responsible for developing training modules, training, identifying the materials needed to support microscopy QA, and improving the performance and maintaining the competence of microscopists. Allocating a small percentage of the national malaria control budget to microscopy QA could yield large benefits through targeted use of costly drugs. 
The second issue is establishment of a separate potent training program for all laboratory workers in the region. National malaria control programs train local microscopists with variable degrees of success. WHO training materials are still widely used, although an update is necessary. , Improving diagnostic accuracy in malaria control systems can be both technically and financially challenging.  Continued supervision and support are essential to ensure sustainability of accurate diagnosis and thereby appropriate treatment.
Limitation of the study: This study did not cover all the peripheral localities of the Red Sea State, only 3 peripheral areas are represented here.
Small number of patients complaining of fever had malaria in Red Sea State with P. falciparum representing (50.2%), followed by P. vivax (43.8%). The best tool for diagnosis of malaria is by doing RDT for any suspected malaria patient, followed by microscopy in RDT-positive cases. Malaria training and quality control programs are highly needed to raise the standard for malaria diagnosis in this area.
Source of Support: None, Conflict of Interest: None