Evaluation of cost-effective total nucleic acids extraction protocols for cultured <it>Mycobacterium tuberculosis</it>; a comparison by PCR amplification of genes associated with drug resistance

Abstract Background The emergence of drug resistant strains of Mycobacterium tuberculosis complex has made the management of tuberculosis difficult. Also, Mycobacterium species has a peculiar cell wall, made of an impermeable complex structure rich in mycolate, making the lyses of its cell difficult. In order to apply a radio-labelled-probe based detection of mutations in selected genes leading to drug resistance, we concede that the evaluation and modifications of nucleic acid extraction protocols that are less sophisticated and less prone to contamination would be useful in the management of tuberculosis in a resource-constrained setting. Findings The average amount of nucleic acids was determined for different extraction treatments. High temperature treatment only, yielded the lowest amount of nucleic acids, i.e. 15.7 ± 3.2 μg. The average amount of nucleic acids obtained with the addition of TE and triton-X100, was 133.7 ± 8.9 μg, while that obtained with the addition of TE only, and TE and SDS were 68.4 ± 22.7 μg and 70.4 ± 20.3 μg respectively. Other treatments yielded 28.8 ± 6.7 μg, 32.5 ± 2.4 μg and 36.9 ± 15.5 μg. The average amount of nucleic acids obtained with high temperature treatment in TE, and that obtained by freezing prior to high temperature treatment, successfully amplified for the genes of interest (rpoB, KatG, rrs). Conclusion We strongly recommend the use of 1× TE buffer, and freezing and heating for improved lysis of cultured M. tuberculosis, and therefore, as an effective method for the preparation of M. tuberculosis nucleic acid useful for PCR.</p

Exploring the influence of sterilisation and storage on some physicochemical properties of coconut <it>(Cocos nucifera L.) </it>water

Abstract Background Fresh coconut (Cocos nucifera L) water is a clear, sterile, colourless, slightly acidic and naturally flavoured drink, mostly consumed in tropical areas. It is a rich source of nutrients and has been used for medical purposes. This study was designed to investigate changes in selected characteristics of coconut water after autoclaving, gamma irradiation and storage. Also, the study was designed for assessing the possibility of measuring the growth of bacterial in fresh, stored or sterilised coconut water using turbidity measurements (at wavelengths between 600 nm and 800 nm) or by dry biomass determinations. Results Portions of coconut water aseptically extracted from the matured fruit, (average pH of 6.33 ± 0.17) were either stored at 4°C, autoclaved at 121°C for 20 min., or irradiated with gamma rays at 5 kGy. Subsequent changes in selected characteristics were determined. Autoclaving, gamma irradiation and long term storage of coconut water at 4°C resulted both in the development of a pale to intense yellow colour and changes in turbidity. After storage, the dry matter content of fresh, autoclaved and irradiated coconut water by 52.0%, 23.5% and 5.0% respectively. There were also significant differences in the UV spectra before and after sterilisation and during the storage of the coconut water. Although changes in total carbohydrates were observed, they were not significant (p > 0.05). Conclusions The enormous differences in the characteristics before and after storage suggests that the use of turbidity and dry biomass measurements for measuring the growth of bacteria in fresh, autoclaved and gamma irradiated coconut water before storage is practicable without any possibility of interference by the innate turbidity, colour and dry matter of the coconut water. However, this is not practicable after storing the coconut waters at 4°C, since there were increases in the turbidity and dry matter of the coconut water to levels that will mask the turbidity of a growing bacteria culture.</p

Potential Bacterial Health Risk Posed to Consumers of Fresh Coconut (Cocos nucifera L.) Water

Coconut (Cocos nucifera L.) water is a refreshing drink consumed mostly directly from the fruit. However, in recent times, consumers in Accra prefer to have it transferred into plastic bags for later consumption; this favours a high risk of bacterial contamination. Since it is rich in nutrient, it may become unwholesome with possible high bacteria loads. However, its use for managing and preventing diarrhoeal diseases and the report that coconut water contains antibacterial proteins, suggests a bacteria growth inhibition potential for it. Therefore, the propensity of fresh coconut water to support the growth of two pathogenic bacteria was studied. Using mostly optical density measurement, and where possible, growth parameters and bacteria loads were estimated for the growth of two gram negative bacteria in fresh, stored and sterilized coconut water, and also in Luria-Bertani (LB) broth as a control. The study revealed that fresh coconut water is a drink favourable for the survival and growth of Escherichia coli, and Klebsiella pneumoniae. It supported the growth of these bacteria, recording lag times of 101.4 ± 1.00 minutes for E. coli and 154.8 ± 0.45 minutes for K. pneumoniae, and high loads of viable cells of ~2.27 × 10 8 cfu/mL and&gt;2.83 × 10 8 cfu/mL at the stationary phase for E. coli and K. pneumoniae respectively. These and other growth parameters in coconut water were comparable to those in Luria-Bertani (LB) broth medium. However, when autoclaved, gamma irradiated or stored at 4˚C for two weeks or more, the growth of these bacteria becomes extremely limited. Fresh coconut water will support the growth of these bacteri

Self-Collected Specimens Revealed a Higher Vaccine- and Non-Vaccine-Type Human Papillomavirus Prevalences in a Cross-Sectional Study in Akuse

Background. Population-specific epidemiologic data on human Papillomavirus infection, which are limited in most of the SubSaharan African countries, are necessary for effective cervical cancer prevention. This study aimed to generate population-specific data on human Papillomavirus infections, and determine which of these, self-collected and provider-collected specimens, gives a higher estimate of the prevalence of human Papillomaviruses, including vaccine and non-vaccine-type human Papillomavirus. Methods. In this cross-sectional study, following a questionnaire-based collection of epidemiological data, self-, and provider-collected specimens, obtained from women 15−65 years of age, were analysed for human Papillomavirus types by a nested-multiplex polymerase chain reaction, and for cervical lesions by Pap testing. HPV data were categorised according to risk type and vaccine types for further analysis. Results. The difference between the overall human Papillomavirus infection prevalences obtained with the self-collected specimens, 43.1% (95% CI of 38.0–51.0%) and that with the provider-collected samples, 23.3% (95% CI of 19.0–31.0%) were significant (p≤0.001). The prevalence of quadrivalent vaccine-type human Papillomaviruses was 12.3% with self-collected specimens, but 6.0% with provider-collected specimens. For the nonavalent vaccine-types, the prevalences were 26.6% and 16.7% respectively. There were multiple infections involving both vaccine-preventable and nonvaccine preventable high-risk human Papillomavirus genotypes. Conclusion. The Akuse subdistrict can, therefore, be said to have a high burden of human Papillomavirus infections, which included nonvaccine types, as detected with both self-collected and provider-collected specimens. These imply that self-collection is to be given a higher consideration as a means for a population-based high-risk human Papillomavirus infections burdens assessment/screening. Additionally, even with a successful implementation of the HPV vaccination, if introduced in Ghana, there is still the need to continue with the screening of women

A tailored within-community specimen collection strategy increased uptake of cervical cancer screening in a cross-sectional study in Ghana

Abstract Background The implementation of cervical cancer screening strategies has reported different rates of success in different countries due to population specific factors that limit women’s participation. We report observations and the development of a community-based specimen collection strategy which resulted from interactions with women in the study communities, following an initial low response to a hospital based cervical cancer screening strategy. Method Women were recruited by a house survey and invited to report at a hospital either within a week or after a week for self and health-personnel specimen collections. However, due to the very low response and subsequent interactions with the women of the communities, another strategy was developed that required recruited women report at a central location within their respective communities for specimen collections at times that did not interfere with their daily routines. Results For specimen collection, of the 156 participants who opted to report after a week at the hospital, 60 (38.5%) reported. Of the 118 participants who opted to report within 1 week at the hospital, 55 (46.6%) reported. Of the 103 participants were invited to report at a specified location within the community, 98 (95.1%) reported. An overall response rate of 60.4% was attained. Almost 89.7% (226 of 253) of the women performed both self and health personnel sample collection. Conclusion The community-based strategy with self-specimen collection and HPV testing holds great potential for increasing women’s participation in cervical cancer screening in Ghana as compared to the hospital based strategy

Unique LCR variations among lineages of HPV16, 18 and 45 isolates from women with normal cervical cytology in Ghana

Abstract Background In addition to being useful for classification, sequence variations of human Papillomavirus (HPV) genotypes have been implicated in differential oncogenic potential and a differential association with the different histological forms of invasive cervical cancer. These associations have also been indicated for HPV genotype lineages and sub-lineages. In order to better understand the potential implications of lineage variation in the occurrence of cervical cancers in Ghana, we studied the lineages of the three most prevalent HPV genotypes among women with normal cytology as baseline to further studies. Methods Of previously collected self- and health personnel-collected cervical specimen, 54, which were positive for HPV16, 18 and 45, were selected and the long control region (LCR) of each HPV genotype was separately amplified by a nested PCR. DNA sequences of 41 isolates obtained with the forward and reverse primers by Sanger sequencing were analysed. Results Nucleotide sequence variations of the HPV16 genotypes were observed at 30 positions within the LCR (7460 – 7840). Of these, 19 were the known variations for the lineages B and C (African lineages), while the other 11 positions had variations unique to the HPV16 isolates of this study. For the HPV18 isolates, the variations were at 35 positions, 22 of which were known variations of Africa lineages and the other 13 were unique variations observed for the isolates obtained in this study (at positions 7799 and 7813). HPV45 isolates had variations at 35 positions and 2 (positions 7114 and 97) were unique to the isolates of this study. Conclusion This study provides the first data on the lineages of HPV 16, 18 and 45 isolates from Ghana. Although the study did not obtain full genome sequence data for a comprehensive comparison with known lineages, these genotypes were predominately of the Africa lineages and had some unique sequence variations at positions that suggest potential oncogenic implications. These data will be useful for comparison with lineages of these genotypes from women with cervical lesion and all the forms of invasive cervical cancers