Patient 1. Index case

A 23-year-old male woodcutter, resident in Vigia del Fuerte (6.35N, 76.53W), was referred to the hospital on 11 April 2001, diagnosed with yellow fever. Vigia del Fuerte is a highly endemic malaria region located in the Atrato River basin with a mean Annual Parasite Index (API) of 72.6 during the past five years. The patient presented 15 days with fever, headache, chills, jaundice, arthralgia, melena, dark urine and bilious vomiting. He was given a heparin lock for administration of I.V. treatment. The patient deteriorated throughout the night and died before thick smear results were available. Peripheral blood thin and thick smears confirmed the presence of P. falciparum asexual forms (>100,000 per μl). A post-mortem confirmed as cause of death complicated malaria. Dengue and yellow fever IgM antibodies were absent. The patient remained in the emergency ward for 18 hours prior to his death.

Patient 2

A 19 year-old male, unemployed, resident in Angelopolis (Antioquia, 6.06°N 75.42°W), a non-malarious area, located 1,900 m a.s.l., attended the emergency service of the hospital on 10^th April 2001, and remained there for 24 hours before being transferred to the Internal Medicine Ward with diagnosis of systemic lupus erythematosus (SLE). In addition to symptoms and signs of SLE, he presented fever on 23 April, and received treatment with fenitoin, dipirone, ampicillin, ranitidine, ceftriazone, methilprednisolone, oxacillin, amikacin and enoxaparine (via an heparin lock). As part of the treatment for his condition, he was administered chloroquine 150 mg daily from 24^th April 2001. He was discharged on 19^th May 2001. Then, on 6^th June 2001, he was hospitalized with a diagnosis of a febrile syndrome. P. falciparum malaria was confirmed on 20^th June 2001 after observation of the parasite in a blood cell count. A thick smear revealed the presence of 14,440 asexual forms per μl in peripheral blood. The patient was administered quinine and sulphadoxine-pyrimethamine with good treatment response.

Patient 3

A 46 year-old male, unemployed, resident of Santa Barbara (Antioquia, 5.57°N 75.91°W), a non-malarious area located 1,800 m a.s.l., was hospitalized in the Internal Medicine Ward on 8^th May with diagnosis of status asthmaticus. He was administered aminophyllin, hydrocortisone, ranitidine, enoxaparine (via a heparin lock) and was discharged on 15^th May 2001. He returned to the hospital on 5^th June 2001 complaining of fever, chills and adynamia and was admitted with diagnosis of toxic hepatitis due to the presence of dark urine, jaundice and hepatomegaly. On 6^th June 2001, diagnosis of complicated malaria was confirmed by observation of the parasite in a blood cell count. Later, the presence of 87,500 trophozoites of P. falciparum per μl was confirmed by a thick smear. He received treatment with quinine, sulphadoxine-pyrimethamine and was discharged on June 23^rd 2001

Patient 4

A 41 year-old male, flower-grower, resident of El Carmen del Viboral (Antioquia, 6.09°N 75.34°W), a non-malarious area located 2,150 m a.s.l.., was admitted directly to the Internal Medicine Ward of the hospital on 7^th May 2001 with superior vena cava syndrome (100% obstruction) and was applied a heparin lock for administration of I.V treatment. The patient was diagnosed with a mediastinal mass, compatible with lymphoma, and received treatment with dexametasone (I.V.) and radiotherapy. On June 9^th 2001, he evidenced fever and on 9^th June 2001, he was diagnosed as complicated malaria by a cell blood count. A thick smear confirmed the presence of 166,440 asexual forms of P. falciparum per μl. He received treatment with quinine, sulphadoxine-pyrimethamine with good treatment response. However, on 1^st July 2001, he died as a consequence of multiple non-malaria related complications.

Entomological captures

Search for Anopheles was carried out by expert personnel from the Secretariat of Health, both within the ward and in the surrounding gardens, around the time of malaria diagnosis of patients 2 and 4. These confirmed the absence of the vector in the hospital area.

Molecular analysis

P. falciparum genomic DNA was extracted from whole blood collected onto filter paper or from paraffin embebbed brain tissue (for the index case). Paraffin was removed from post-morten material using xylene, followed by 2 washes with 100% ethanol. DNA was isolated by proteinase K digestion, followed by four rounds of phenol-chloroform extraction. Purified DNA was stored at -20°C until amplification.

The Region 2 of MSP-1 and the central region of MSP-2 were amplified by a nested Polymerase Chain Reaction (PCR); the region RII of GLURP was amplified by a semi-nested PCR [1]. Products obtained after the first PCR were amplified using specific primers for region 2 of MSP-1 corresponding to MAD20, K1 and RO33 allelic families, and FC27 and IC-1 for the central region of MSP-2. Briefly, PCR was carried out in a total volume of 20 μL, containing 10 mm Tris-HCl (pH 9,0 at 25°C), 50 mm KCl and 0,1% Triton X-100, 125 mM dNTPs, 0,4 units Taq DNA polymerase (Promega, Madison, WI), 1,6 mM MgCl2 and 125 nM of each primer. Initial denaturation was 5 min at 95°C, 1 min at 94°C, 2 min at 58°C annealing (all first PCR reactions and second reaction for GLURP) or at 61°C (for all the second reactions of MSP-1 and 2). This was followed by extension for 2 minutes at 72°C. This first reaction underwent 25 amplification cycles and the second 30 cycles. Positive (strains HB3, K1 and RO33) and negative controls (healthy individuals), were included.

Products were electrophoresed on an agarose (MetaPhor) gel (2,5% for MSP-1 and 2% for MSP-2 and GLURP), stained with ethidium bromide and visualized under ultraviolet light. Size analysis of the amplified fragments revealed identical pattern distribution for all the examined markers assayed, confirming the presence of P. falciparum infection by matching strains in all 4 patients (Fig 1).