Report of autochthonous cases of localized cutaneous leishmaniasis caused by Leishmania (Leishmania) mexicana in vulnerable, susceptible areas of Southeastern Mexico

ABSTRACT Localized cutaneous leishmaniasis (LCL) is an endemic disease in several Mexican States with the main endemic areas located in the South-Southeast region of the country, where 90% of Leishmania (Leishmania) mexicana cases are registered. The Southeast region is located in the Yucatan Peninsula, including Campeche, Quintana Roo and Yucatan States. Campeche and Quintana Roo register more than 60% of the cases in the country each year, while in Yucatan the reports are of imported cases due to residents traveling to endemic areas. However, since 2015, autochthonous cases have been diagnosed by health authorities in municipalities with no previous transmission records. We aimed to identify Leishmania parasite species involved in autochthonous cases by means of the PCR technique. The present study included 13 autochthonous cases of LCL with clinical and parasitological diagnoses during 2018 and 2019 by health authorities, without specific identification of the causal agent. Tissue samples were taken by scraping the margins of active lesions and then they were spotted onto an FTATM Elute Microcard. Next, DNA was eluted and used for PCR amplification of specific Leishmania genus and L. (L.) mexicana species-specific fragments. Molecular analysis showed evidence that L. (L.) mexicana was the causal agent of LCL in 12 of the 13 patients; in one patient, PCR was not performed due to the patient's refusal to participate in the study. Identifying Leishmania species that cause LCL is necessary to define efficient treatment schemes and control strategies for the disease in vulnerable and susceptible areas of the Yucatan State's municipalities.

Development of a species-specific PCR assay for authentication of Agkistrodon acutus based on mitochondrial cytochrome b gene

BACKGROUND: Agkistrodon acutus, a traditional Chinese medicine, clinically used in the treatment of rheumatism, tumor, and cardiovascular and cerebrovascular diseases. Due to the unique medicinal value and the difficulty of artificial breeding of Agkistrodon acutus, the supply of Agkistrodon acutus on the market exceeds the demand, and a large number of its adulterants are found on the market. In this study, the cytb gene sequences of Agkistrodon acutus and 9 snakes were compared and analyzed, specific primers were designed, and specific PCR methods were established to detect Agkistrodon acutus medicinal samples on the market. RESULTS: This method was successfully applied to distinguish the snake from other adulterated species, and tested 18 Agkistrodon acutus samples randomly purchased from six cities. Twelve samples were counterfeit and six were genuine. The standard reference material of Agkistrodon acutus was cloned by molecular cloning and sequencing, and the gene sequence difference with other species was significant. It shows that the region could be used as the fingerprint region of the target species. CONCLUSIONS: The proposed method can be used as a species-specific marker and can be highly distinguished from other adulterated snake species, which is helpful to effectively avoid the problem of false sale of Agkistrodon acutus.

Distribution of Meloidogyne species in carrot in Brazil / Distribuição de espécies de Meloidogyne em cenoura no Brasil

ABSTRACT: Root-knot nematodes (RKN - Meloidogyne spp.) are one of the most serious threats to carrot production worldwide. In Brazil, carrots are grown throughout the year, and economic losses due to RKN are reported. Since little is known on the distribution of RKN species in carrot fields in Brazil, we collected plant and soil samples from 35 fields across six states. Based on the morphology of perineal patterns, esterase phenotypes and species-specific PCR, three Meloidogyne species were identified: 60% of the fields were infested with Meloidogyne incognita, M. javanica was reported in 42.9% of the areas, whereas M. hapla was detected in 17.1% of carrot fields. Mixed populations were reported in 20% of the areas with a predominance of M. incognita + M. javanica. The combination of morphological, biochemical, and molecular techniques is a useful approach to identify RKN species.

RESUMO: Os nematoides-das-galhas (RKN - Meloidogyne spp.) são uma das mais sérias ameaças à produção de cenoura no mundo. No Brasil, as cenouras são cultivadas ao longo do ano, e as perdas econômicas devido à RKN são frequentemente relatadas. Como pouco se sabe sobre a distribuição de espécies RKN em campos de cenoura no Brasil, coletamos amostras de plantas e solo de 35 campos em seis estados. Baseado na morfologia do padrão perineal, fenótipos de esterase e/ou PCR espécie-específica, três espécies de Meloidogyne foram identificadas: 60% dos campos estavam infestados por Meloidogyne incognita, M. javanica foi encontrada em 42,9% das áreas, enquanto M. hapla foi detectada em 17,1% dos campos de cenoura. Populações mistas foram encontradas em 20% das áreas, com predominância de M. incognita + M. javanica. A combinação de técnicas morfológicas, bioquímicas e moleculares é uma abordagem útil para identificar espécies de RKN.

Molecular markers based upon whole chloroplast genomes and identifying alpine Gentiana waltonii and G. lhassica (Gentianaceae) / 药学学报

As two original plants of Tibetan herb Jieji, Gentiana waltonii Burk. and Gentiana lhassica Burk. belong to Section Cruciata of Gentiana, Gentianaceae. Here, we report on whole chloroplast genome sequences in the alpine species, respectively, and the features of plastomes were investigated. The plastome of G. waltonii is 148 705 bp long (148 652 bp in G. lhassica) and encodes 112 genes, including 78 protein-coding genes, 30 transfer RNA genes, and 4 ribosomal RNA genes. Two pseudogenes, namely ψrps16 and ψinfA, were found in plastomes. In addition, two novel loci were detected, and a species-specific polymerase chain reaction assay was developed for differentiating G. waltonii and G. lhassica from 10 alpine species in Section Cruciata. Gentiana. Our study provides basic data for identifying Tibetan herbs, alpine species conservation and molecular phylogenetic studies of Gentiana and Gentianaceae.

Development of Streptococcus sanguinis-, Streptococcus parasanguinis-, and Streptococcus gordonii-PCR Primers Based on the Nucleotide Sequences of Species-specific DNA Probes Screened by Inverted Dot Blot Hybridization

The objective of this study was to develop PCR primers that are specific for Streptococcus sanguinis, Streptococcus parasanguinis, and Streptococcus gordonii. We designed the S. sanguinis-, S. parasanguinis-, and S. gordonii-specific primers, Ssa21-F3/Ssa21-R2, Spa17-F/Spa17-R, and Sgo41-F1/Sgo41-R1 respectively, based on the nucleotide sequences of the Ssa21, Spa17, and Sgo41 DNA probes that were screened using inverted dot blot hybridization (IDBH). The species-specificity of these primers was assessed against 43 strains of mitis group streptococci, including clinical strains of S. sanguinis, S. parasanguinis, and S. gordonii. The resulting PCR data revealed that species-specific amplicons had been obtained from all strains of the target species tested, and that none of these amplicons occurred in any other strains from other species. These results suggest that the Ssa21-F3/Ssa21-R2, Spa17-F/Spa17-R, and Sgo41-F1/Sgo41-R1 primers may be useful in detecting S. sanguinis, S. parasanguinis, and S. gordonii at the species level, respectively.

Discriminative PCR of Bordetella pertussis from closely related Bordetella species using 16S rDNA Gene / 감염과화학요법

BACKGROUND: Polymerase-chain reaction (PCR) detection is useful to diagnosis of pertussis at initial stage because the growth rate of Bordetella pertussis (B. pertussis) is relatively slow. Currently, the primer set for the insertion sequence IS481 (BP primer) is used widely for PCR detection of B. pertussis. However, the cross-reactivity of BP primer set with Bordetella holmesii (B. holmesii) was reported recently. Therefore, discrimination of B. pertussis and B. holmesii is needed in PCR step. For this reason, we developed new primer sets based on 16S rDNA sequence for diagnostic use and estimated the efficiency of these new primer sets. MATERIALS AND METHODS: The specific PCR primers were designed from the aligned sequence matrix of 16S rDNA genes of various Bordetella species. The specificity of designed primers were estimated using clinically important 4 Bordetella species, B. pertussis, B. holmesii, Bordetella parapertussis (B. parapertussis) and Bordetella bronchiseptica (B. bronchiseptica). The sensitivity to B. pertussis of designed primers was also estimated and compared with BP primer set. RESULTS: As the results, the developed new primer set successfully distinguished B. pertussis and other Bordetella species containing B. holmesii. In the sensitivity assay, the detectable limits of 16S-F2/16S-R1 primer set for B. pertussis were revealed as 5 pg of genomic DNA and 105 cells/mL of cell suspension. In addition to these, identical results between BP with primer and new primer were obtained in clinical samples. CONCLUSION: In this study, the specific primer set for B. pertussis was developed based on 16S rDNA sequence and this primer set did not show cross-reactivity to B. holmesii. In addition to these, the applicability of this primer set to the clinical specimens was also confirmed.

Discriminative PCR of Bordetella pertussis from closely related Bordetella species using 16S rDNA Gene / 감염과화학요법

BACKGROUND: Polymerase-chain reaction (PCR) detection is useful to diagnosis of pertussis at initial stage because the growth rate of Bordetella pertussis (B. pertussis) is relatively slow. Currently, the primer set for the insertion sequence IS481 (BP primer) is used widely for PCR detection of B. pertussis. However, the cross-reactivity of BP primer set with Bordetella holmesii (B. holmesii) was reported recently. Therefore, discrimination of B. pertussis and B. holmesii is needed in PCR step. For this reason, we developed new primer sets based on 16S rDNA sequence for diagnostic use and estimated the efficiency of these new primer sets. MATERIALS AND METHODS: The specific PCR primers were designed from the aligned sequence matrix of 16S rDNA genes of various Bordetella species. The specificity of designed primers were estimated using clinically important 4 Bordetella species, B. pertussis, B. holmesii, Bordetella parapertussis (B. parapertussis) and Bordetella bronchiseptica (B. bronchiseptica). The sensitivity to B. pertussis of designed primers was also estimated and compared with BP primer set. RESULTS: As the results, the developed new primer set successfully distinguished B. pertussis and other Bordetella species containing B. holmesii. In the sensitivity assay, the detectable limits of 16S-F2/16S-R1 primer set for B. pertussis were revealed as 5 pg of genomic DNA and 105 cells/mL of cell suspension. In addition to these, identical results between BP with primer and new primer were obtained in clinical samples. CONCLUSION: In this study, the specific primer set for B. pertussis was developed based on 16S rDNA sequence and this primer set did not show cross-reactivity to B. holmesii. In addition to these, the applicability of this primer set to the clinical specimens was also confirmed.

Development of Species-specific Primers for Rapid Detection of Phellinus linteus and P. baumii

Genus Phellinus taxonomically belongs to Aphyllophorales and some species of this genus have been used as a medicinal ingredients and Indian folk medicines. Especially, P. linteus and morphological-related species are well-known medicinal fungi that have various biological activities such as humoral and cell-mediated, anti-mutagenic, and anti-cancer activities. However, little is known about the rapid detection for complex Phellinus species. Therefore, this study was carried out to develop specific primers for the rapid detection of P. linteus and other related species. Designing the species-specific primers was done based on internal transcribed spacer sequence data. Each primer set detected specifically P. linteus (PL2/PL5R) and P. baumii (PB1/PB4R). These primer sets could be useful for the rapid detection of specific-species among unidentified Phellinus species. Moreover, restriction fragment length polymorphism analysis of the ITS region with HaeIII was also useful for clarifying the relationship between each 5 Phellinus species.