A sample of Aedes aegypti L. from Santiago de Cuba with a high level of deltamethrin resistance (113.7 x at the 50% lethal concentration [LC50]), was subjected to deltamethrin selection to determine the capacity of this population to evolve higher resistance under intensive laboratory selection pressure, to characterize that resistance, to attempt to identify some of the mechanisms involved, and to use it as a reference strain for future molecular research. High resistance developed after 12 generations of selection (1,425 x). After selection for 12 generations with deltamethrin, the Santiago de Cuba colony (SAN-F12) showed little or no cross-resistance to the organophosphates evaluated, but high cross-resistance was observed for all the pyrethroids in larvae from this strain: lambdacyhalothrin (197.5 x), cypermethrin (45 x), and cyfluthrin (41.2 x). Adult bioassays reveal that a SAN-F12 strain was resistant to the pyrethroid and the organochlorine dichlorodiphenyltrichloroethane (DDT). Synergism tests implicated detoxifying esterase or glutathione S-transferase (GST) and monooxygenase in pyrethroid resistance. Biochemical tests reveal that acetylcholinesterase was not involved in deltamethrin resistance. The frequency of GST enzyme increased from 0.43 in Santiago de Cuba to 0.88 in SAN-F12. Esterase frequency increased from 0.12 in Santiago de Cuba to 0.63 in SAN-F6 and it diminished to 0.38 in SAN-F12. The polyacrylamide gel electrophoresis and inhibition study suggests the presence of elevated esterase activity not associated with pyrethroid resistance. The presence of both DDT and pyrethroid resistance in the SAN-F12 strain suggests the presence of a knockdown (Kdr)-type resistance mechanism, although the frequency of this mechanism was low. Resistance to deltamethrin could be associated with esterase or GST mechanisms, and more investigation is required. This information contributes to the improvement of resistance management strategies in the Cuban Ae. aegypti control program.

Genotypes of two different loci of the Pneumocystis jirovecii mitochondrial gene were studied in specimens from a total of 75 Pneumocystis pneumonia patients in Spain, France and Cuba. A new genotype of the mitochondrial small subunit rRNA gene of P. jirovecii (160A/196T) was identified, which was revealed to be the most common in these three countries, especially in Cuba where its proportion reached 93.8%. Our data imply that the new genotype might be circulating worldwide and also suggests that the distribution of P. jirovecii genotypes could be narrower in islands such as Cuba.

Pneumocystis jirovecii causes severe interstitial pneumonia (PcP) in immunosuppressed patients. This multicentre study assessed the distribution frequencies of epidemiologically relevant genetic markers of P. jirovecii in different geographic populations from Portugal, the USA, Spain, Cuba and Mozambique, and the relationship between the molecular data and the geographical and clinical information, based on a multifactorial approach. The high-throughput typing strategy for P. jirovecii characterization consisted of DNA pooling using quantitative real-time PCR followed by multiplex-PCR/single base extension. The frequencies of relevant P. jirovecii single nucleotide polymorphisms (mt85, SOD110, SOD215, DHFR312, DHPS165 and DHPS171) encoded at four loci were estimated in ten DNA pooled samples representing a total of 182 individual samples. Putative multilocus genotypes of P. jirovecii were shown to be clustered due to geographic differences but were also dependent on clinical characteristics of the populations studied. The haplotype DHFR312T/SOD110C/SOD215T was associated with severe AIDS-related PcP and high P. jirovecii burdens. The frequencies of this genetic variant of P. jirovecii were significantly higher in patients with AIDS-related PcP from Portugal and the USA than in the colonized patients from Portugal, and Spain, and children infected with P. jirovecii from Cuba or Mozambique, highlighting the importance of this haplotype, apparently associated with the severity of the disease and specific clinical groups. Patients from the USA and Mozambique showed higher rates of DHPS mutants, which may suggest the circulation of P. jirovecii organisms potentially related with trimethoprim-sulfamethoxazole resistance in those geographical regions. This report assessed the worldwide distribution of P. jirovecii haplotypes and their epidemiological impact in distinct geographic and clinical populations.

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e This study describes the prevalence and genotype distribution of Pneumocystis jirovecii obtained from nasopharyngeal (NP) swabs from immunocompetent Cuban infants and toddlers with whooping cough (WC). A total of 163 NP swabs from 163 young Cuban children with WC who were admitted to the respiratory care units at two pediatric centers were studied. The prevalence of the organism was determined by a quantitative PCR (qPCR) assay targeting the P. jirovecii mitochondrial large subunit (mtLSU) rRNA gene. Genotypes were identified by direct sequencing of mtLSU ribosomal DNA (rDNA) and restriction fragment length polymorphism (RFLP) analysis of the dihydropteroate synthase (DHPS) gene amplicons. qPCR detected P. jirovecii DNA in 48/163 (29.4%) samples. mtLSU rDNA sequence analysis revealed the presence of three different genotypes in the population. Genotype 2 was most common (48%), followed in prevalence by genotypes 1 (23%) and 3 (19%); mixed-genotype infections were seen in 10% of the cases. RFLP analysis of DHPS PCR products revealed four genotypes, 18% of which were associated with resistance to sulfa drugs. Only contact with coughers (prevalence ratio [PR], 3.51 [95% confidence interval {CI}, 1.79 to 6.87]; P ‫؍‬ 0.000) and exposure to tobacco smoke (PR, 1.82 [95% CI, 1.14 to 2.92]; P ‫؍‬ 0.009) were statistically associated with being colonized by P. jirovecii. The prevalence of P. jirovecii in infants and toddlers with WC and the genotyping results provide evidence that this population represents a potential reservoir and transmission source of P. jirovecii.