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Increasing resistance, recrudescences, and treatment failure have led to the replacement of chloroquine with the combination of pyrimethamine (PYR) and sulfadoxine (SDX) as the first-line antimalarial drugs for treatment of uncomplicated Plasmodium falciparum malaria in several areas where this disease is endemic. The development of resistance to PYR-SDX is favored by incomplete treatment courses or by subtherapeutic levels in plasma. PYR-SDX resistance has been associated with several single-nucleotide polymorphisms (SNPs) in the P. falciparum dihydrofolate reductase ( pfdhfr ) and the P. falciparum dihydropteroate synthetase ( pfdhps ) genes. We have established assays based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that conveniently allow the identification of SNPs associated with PYR resistance. Variants occurring at codon positions 16, 51, 59, and 108 of the pfdhfr gene were analyzed by MALDI-TOF MS in synthetic oligonucleotides to determine the detection threshold. In addition, 63 blood samples from subjects with P. falciparum parasitemia of various degrees were analyzed. The results were compared to those obtained by DNA sequencing of the respective gene fragment. The results of MALDI-TOF MS and DNA sequencing were consistent in 40 samples. In 23 samples two or three pfdhfr variants were detected by MALDI-TOF assays, whereas DNA-sequencing revealed one variant only. Simultaneous detection of two different mutations by biplex assays was, in principle, feasible. As demonstrated by the example of PYR resistance, MALDI-TOF MS allows for rapid and automated high-throughput assessment of drug sensitivity in P. falciparum malaria.

The surface M protein of group A streptococci binds factor H, a regulatory protein of the alternative complement pathway, which may contribute to the antiphagocytic activity of the M molecules. To locate the factor H binding domain in the alpha-helical coiled-coil structure of the M molecule, the M protein was cleaved with pepsin at pH 5.8, which separates the molecule approximately in half. Western blot (immunoblot), amino acid sequence, and mass spectrometric analyses revealed that factor H bound to a 14.6-kDa C-terminal fragment of the M molecule. Competitive inhibition of factor H binding to the 14.6-kDa fragment with M protein peptides localized the binding site to amino acids 256 to 292. This segment is located within the surface-exposed region of the M6 protein, identified as the C-repeat region, whose sequence is conserved among heterologous M and M-like molecules. These studies also identified a second pepsin-susceptible site with the sequence ELAK located within the cell wall-associated region of the M molecule.

M protein, the major virulence factor of group A streptococci, has antiopsonic activity in that it inhibits activation of the alternative complement pathway on the streptococcal surface. Two properties of M protein have been claimed to account for the inhibitory activity, namely, (i) its binding affinity for complement factor H, which is an inhibitor of alternative pathway activation, and (ii) its high binding affinity for fibrinogen. We have recently shown that fibrinogen, like M protein, inhibits alternative pathway activation by possessing binding affinity for factor H. Here we report that fibrinogen effectively competes with factor H for binding to M protein but retains its own binding affinity for factor H. The presence of fibrinogen did not significantly affect alternative pathway inhibition on the streptococcal surface.