In vitro adaptation of Plasmodium falciparum reveal variations in cultivability

Abstract

Background: Culture-adapted Plasmodium falciparum parasites can offer deeper understanding of geographic variations in drug resistance, pathogenesis and immune evasion. To help ground population-based calculations and inferences from culture-adapted parasites, the complete range of parasites from a study area must be well represented in any collection. To this end, standardized adaptation methods and determinants of successful in vitro adaption were sought.

Methods: Venous blood was collected from 33 P. falciparum-infected individuals at Goa Medical College and Hospital (Bambolim, Goa, India). Culture variables such as whole blood versus washed blood, heat-inactivated plasma versus Albumax, and different starting haematocrit levels were tested on fresh blood samples from patients. In vitro adaptation was considered successful when two four-fold or greater increases in parasitaemia were observed within, at most, 33 days of attempted culture. Subsequently, parasites from the same patients, which were originally cryopreserved following blood draw, were retested for adaptability for 45 days using identical host red blood cells (RBCs) and culture media.

Results: At a new endemic area research site, ~65% of tested patient samples, with varied patient history and clinical presentation, were successfully culture-adapted immediately after blood collection. Cultures set up at 1% haematocrit and 0.5% Albumax adapted most rapidly, but no single test condition was uniformly fatal to culture adaptation. Success was not limited by low patient parasitaemia nor by patient age. Some parasites emerged even after significant delays in sample processing and even after initiation of treatment with anti-malarials. When 'day 0' cryopreserved samples were retested in parallel many months later using identical host RBCs and media, speed to adaptation appeared to be an intrinsic property of the parasites collected from individual patients.

Conclusions: Culture adaptation of P. falciparum in a field setting is formally shown to be robust. Parasites were found to have intrinsic variations in adaptability to culture conditions, with some lines requiring longer attempt periods for successful adaptation. Quantitative approaches described here can help describe phenotypic diversity of field parasite collections with precision. This is expected to improve population-based extrapolations of findings from field-derived fresh culture-adapted parasites to broader questions of public health importance.

Fig. 1

Workflow for testing adaptability of Plasmodium falciparum parasites from patients. Between 3 and 6 ml of blood was collected from each enrolled patient infected with P. falciparum or mixed co-infection with P. vivax. For direct culture (left path), aliquots of infected blood were used to establish 8 cultures under different conditions (See “Methods” and Table 1). For the adaptation from cryopreserved samples (right path), two independent researchers each thawed cryogenically preserved patient isolate samples in duplicate. When two cultures reached 0.5–1.0 %, two growth tests were conducted. Parasite cultures were maintained for as long as 35 days for adaptation from freshly drawn blood and as long as 45 days to allow adaptation from cryopreserved blood

Fig. 2

Time to adaptation is not affected by growth medium. For cultures initiated for adaptation from fresh blood draws, pairwise comparisons between all possible combinations of protocols showed no significant difference in time to adaptation between HI-plasma- vs Albumax-supplemented media nor between unwashed (whole blood) vs washed pRBCs (p > 0.1). All haematocrits are treated together in this data (Table 1)

Fig. 3

Time to adaptation varied predictively and reproducibly between different patient isolates. a A comparison of pairwise time-to-adaptation for parasites from direct blood draws versus from cryopreservation. Parasite samples that were subjected to immediate adaptation after a blood draw showed the same ranked-tendencies compared to cryopreserved immediately after blood draw from the patients, when tested together months later in identical media and identical host RBCs, the isolates showed roughly the same ranking in ease to adaptation. The fastest adapting parasites from some fresh patient blood draws (to the left) adapted as quickly as 8 days in culture (e.g., CA1 and CA14), others (e.g., CA15, CA16, CA19) took 15–20 days in culture, and yet others (NA4 and NA11) did not adapt at all. For this work, blinded, duplicate samples were adapted by each of two different scientists in the laboratory. Samples with a history of anti-malarial treatment or prolonged exposure to room temperature before sample processing were left out (see “Results”). b Cryopreserved parasites from individual patient isolates show large but reproducible variations in time to adaptation. For each cryopreserved patient parasite isolate, the vertical line inside a box represents the mean number of days to adaptation. The box around the vertical line represents the full spread of days to adaptation for that parasite isolate. The raw data is shown in Table 2

Fig. 4

Time to adaptation was shorter in parasites derived from older patients than in parasites from younger donors. a Patient samples adapted straight from fresh blood draws showed faster time to adaptation in patients above the age of 32 than below the age of 32. b Patient samples adapted after cryopreservation had a less significant relationship between speed of adaptation and the age of the donor

Fig. 5

Time to adaptation was not related to patient parasitaemia. a Patient samples adapted from fresh blood draws showed roughly the same time to adaptation 8–22 days, even as parasitaemia ranged from <0.1 in 3 samples to >9 % in 3 samples. b After cryopreservation, low parasitaemia samples showed longer time to adaptation than fresh samples when starting parasitaemia was less than 2 %. This was presumably due to fragility of parasitized RBC. Recovery and adaptation was robust at parasitaemia above 2 %