Chahbahar district has been selected as the study area for the following reasons:
Inhabitants are from different ethnic and racial groups (Bluch, Fars, Afghan, Pakistani and etc.), some with family members living 200–300 km away in the Bluchistan Province of Iran and the Bluchistan Province of Pakistan.
Movement of people from different parts of Iran, Pakistan, Afghanistan and the other side of the Oman sea to Chahbahar for holidays, job-hunting and financial activities may affect parasite populations and cause the re-introduction of malaria into areas, where an interruption of transmission had been achieved before (i.e. in the northern, central and western).
Presence of some P. falciparum cases with no response to chloroquine treatment in Chahbahar district.
To date the situation in south-eastern Iran is quite serious, because of the proximity to Afghanistan, a country with disrupted health systems.
Microscopy has historically been the mainstay of the diagnosis of malaria. A clinical diagnosis of malaria currently depends on the visualization of parasites by light microscopy of Giemsa-stained thick and thin blood smears. This procedure is cheap and simple, but it is a labour intensive procedure and requires well-trained personnel . Many studies have demonstrated the greater sensitivity and specificity of PCR compared to thick blood films. The detection of low P. vivax and P. falciparum parasitaemia by PCR, at levels undetectable by microscopy, has been reported by Brown et al. , Sethabutr et al. , Snounou et al. , Wataya et al. , Khoo et al. , Black et al. , Roper et al.  and Singh et al. . The present study between microscopy and nested PCR assay showed that the results obtained by PCR were equivalent or superior to those obtained by microscopy, in that all microscopy-positive samples were positive by PCR. In addition, the PCR test was able to detect mixed infections that were missed by microscopy. This may be due to the tendency to one species to be dominant over other species .
With the spread of parasite resistance to antimalarial drugs in Sistan and Bluchistan Province and the increasing difficulty in controlling malaria in these areas, it is important to diagnose malaria accurately and to treat it correctly. Microscopic observation of parasites stained with Giemsa in thick smears is an inexpensive and simple method that is still used in these areas with malaria transmission and where the diagnosis of malaria is part of primary health care. Several malaria infections from endemic countries are subpatent, with very low parasitaemia, and our results also showed this has occurred in our study area. The problem of population migration, together with the possibility of tourists and professionals travelling to areas at risk for malaria, has increased the number of cases in areas in which malaria transmission was low or previously eradicated. In these cases, an accurate malaria diagnosis is very important so that a possible recrudescence after an incorrect treatment of infected individuals can be avoided.
Mixed infections with asexual blood forms of P. falciparum and P. vivax are well described but relatively uncommon compared to single species infections. Shute (1951) described the frequency of mixed infections as less than 1% among the hundreds of British troops he examined in southern Italy during 1943 . Traditionally, P. falciparum has been thought to inhibit the parasitaemia of P. vivax . In contrast, there are several lines of evidence suggesting that P. vivax may have a suppressive effect on P. falciparum. James , in his classic review of studies of induced malaria was impressed that P. vivax was the predominant species. In their studies of induced malaria, Boyd & Kitchen  often used small doses of quinine which has long been known to have a greater suppressive effect on P. vivax than on P. falciparum . In three instances where P. vivax parasitaemia rose and no drug was administered, asexual P. falciparum parasitaemia fell to submicroscopic levels . Drug selection for the treatment of malaria depends on species of malaria present. Delayed or missed diagnosis of falciparum malaria increases the risk of complicated or severe disease, which may be fatal, especially in non-immunes, and many isolates of P. falciparum are chloroquine resistant and thus would not be eradicated by the standard treatment for P. vivax. When parasite levels are very low and in the detection of mixed species infections, the information obtained by microscopy is restricted, and in some cases biased, by the inability to devote the necessary amount of time to the examination of blood smears. A missed diagnosis of P. vivax concurrent with P. falciparum is more problematic since these species could cause relapses, thereby compounding morbidity. Because of negative microscopical diagnosis untreated patients may be carriers of the malaria parasites in these particular areas. The number of patients who had travelled to Pakistan, Afghanistan and other parts of Sistan and Bluchistan Province were high, with the risk of introducing new isolates (including drug resistant parasites) from neighbouring countries.