Updated emm-typing protocol for Streptococcus pyogenes.

OBJECTIVES: PCR-based typing of the emm gene Streptococcus pyogenes often results in the amplification of multiple bands. This has resulted in the misclassification of strains into types based on non-emm gene sequences. We aimed to improve the specificity of the emm typing PCR reaction using a primer called CDC3, the sequence for which has been previously used to identify emm genes in silico. METHODS: The proposed primer CDC3 was validated in silico from a global database of 1688 GAS genomes and in vitro with 32 isolates. PCR reactions were performed on genomic DNA from each isolate, using the published CDC1 forward primer with the CDC2 reverse primer or the new CDC3 reverse primer. The products were examined by gel electrophoresis, and representative PCR products were sequenced. RESULTS: In 1688 S. pyogenes genomes, the previous CDC2 reverse primer annealed in silico in 1671 emm genes and also in 2109 non emm genes in close proximity, whereas the new CDC3 primer annealed in 1669 emm genes only. The remaining 19 genes without a CDC3 binding site were chimeric emm genes. The PCR pair CDC1+CDC3 produced a single band at appropriate molecular weight in all 32 isolates tested, while the CDC1+CDC2 pair produced more than one band in 13 of 32 isolates (40%). CONCLUSIONS: The new CDC3 primer is more specific for emm genes than the previous CDC2 primer and represents a simple solution to reduce the potential for mistyping S. pyogenes strains.

Bacteremia in Childhood Life-Threatening Infections in Urban Gambia: EUCLIDS in West Africa.

BACKGROUND: The limited availability of microbiology services in sub-Saharan Africa impedes accurate diagnosis of bacterial pathogens and understanding of trends in prevalence and antibiotic sensitivities. We aimed to characterize bacteremia among hospitalized children in The Gambia and to identify factors associated with bacteremia and mortality. METHODS: We prospectively studied children presenting with suspected severe infection to 2 urban hospitals in The Gambia, between January 2013 and September 2015. Demographic and anthropometric data, clinical features, management, and blood culture results were documented. Urine screens for antibiotic activity were performed in a subset of participants. RESULTS: Of 411 children enrolled (median age, 29 months; interquartile range, 11-82), 79.5% (325 of 409) reported prehospital antibiotic use. Antimicrobial activity by urinary screen for antibiotic activity was detected in 70.8% (n = 80 of 113). Sixty-six bacterial pathogens were identified in 65 (15.8%) participants and Staphylococcus aureus predominated. Gram-positive organisms were more commonly identified than Gram-negative (P < .01). Antibiotic resistance against first-line antimicrobials (ampicillin and gentamicin) was common among Gram-negative bacteria (39%; range, 25%-100%). Factors significantly associated with bacteremia included the following: gender, hydration status, musculoskeletal examination findings, admission to the Medical Research Council The Gambia at London School of Hygiene & Tropical Medicine hospital, and meeting sepsis criteria. Those associated with increased mortality were presence of a comorbidity, clinical pallor, tachypnea, and altered consciousness. Tachycardia was associated with reduced mortality. CONCLUSIONS: The bacteremia rate in children with suspected childhood life-threatening infectious diseases in The Gambia is high. The pattern of pathogen prevalence and antimicrobial resistance has changed over time compared with previous studies illustrating the importance of robust bacterial surveillance programs in resource-limited settings.

Risk factors for Group B Streptococcus colonisation and disease in Gambian women and their infants.

OBJECTIVES: To determine risk factors for GBS colonisation in Gambian mothers and in their infants from birth to day 60-89 of age. METHODS: Swabs and breastmilk from mothers/infant pairs were collected and cultured on selective agar. Negative samples were analysed for GBS DNA via real-time PCR. Positive isolates were serotyped using multiplex PCR and gel-agarose electrophoresis. RESULTS: Seven hundred and fifty women/infant pairs were recruited. 253 women (33.7%) were GBS-colonised at delivery. The predominant serotypes were: V (55%), II (16%), III (10%), Ia (8%) and Ib (8%). 186 infants were colonised (24.8%) at birth, 181 (24.1%) at 6 days and 96 at day 60-89 (14%). Infants born before 34 weeks of gestation and to women with rectovaginal and breastmilk colonisation at delivery had increased odds of GBS colonisation at birth. Season of birth was associated with increased odds of persistent infant GBS colonisation (dry season vs. wet season AOR 2.9; 95% CI 1.6-5.2). CONCLUSION: GBS colonisation is common in Gambian women at delivery and in their infants to day 60-89 and is dominated by serotype V. In addition to maternal colonisation, breastmilk and season of birth are important risk factors for infant GBS colonisation.

First Report of Rice yellow mottle virus in Rice in The Gambia.

Rice yellow mottle virus (RYMV) of the genus Sobemovirus is a major biotic constraint to rice (Oryza sativa) production in Africa. First reported in Kenya during 1966, RYMV was later found in most countries in Africa where rice is grown (1). In countries in westernmost Africa (The Gambia, Guinea-Bissau, Mauritania, and Senegal), plants with leaf yellowing and mottling symptoms were observed, but RYMV was never isolated. Rice is the staple food in The Gambia. In 2006, four samples were collected from local rice varieties in the Kuntaur Region in the center of The Gambia. Mechanical inoculation with leaf extracts from all samples caused typical yellow mottle symptoms on the susceptible rice varieties BG90-2, Bouaké 189, and IR64. RYMV was detected in the four samples collected by ELISA with polyclonal antisera (2). The 720-nt coat protein gene was amplified for each isolate by reverse-transcriptase-PCR with primers 5'-CAAAGATGGCCAGGAA-3' (sense) and 5'-CTCCCCCACCCATCCCGAGAATT-3' (antisense) (2). The RT-PCR products were directly sequenced (EMBL Accession Nos. AM765810, AM765811, AM765812, and AM765813) and then aligned using ClustalW with a pool of RYMV coat protein sequences from West African isolates (EMBL Accession Nos. AJ279905, AJ279901, AJ885137, AJ885124, and AJ279935). Phylogenetic reconstruction by maximum-likelihood with PAUP indicated that the isolates from The Gambia formed a monophyletic group with over 97% nucleotide identity and are closely related to isolates of other countries in West Africa (Burkina Faso, Côte d'Ivoire, Guinea, Mali, and Sierra-Leone) with 91 to 94% identity. Detection of RYMV in The Gambia indicates that RYMV is present in westernmost Africa, which is referred to as the 'rice belt' of Africa, and shows that RYMV is widely distributed from eastern Africa (Tanzania) to the western part of the continent. References: (1) N. K. Kouassi et al. Plant Dis. 89:124, 2005. (2) A. Pinel et al. Arch. Virol. 145:1621, 2000.