Integrated microscale immiscible phase extraction and isothermal amplification for colorimetric detection of Neisseria gonorrhoeae.

Gonorrhea is the second most common sexually transmitted infection (STI) with around 87 million cases worldwide estimated in 2016 by the World Health Organization. With over half of the cases being asymptomatic, potential life-threatening complications and increasing numbers of drug-resistant strains, routine monitoring of prevalence and incidence of infections are key preventive measures. Whilst gold standard qPCR tests have excellent accuracy, they are neither affordable nor accessible in low-resource settings. In this study, we developed a lab-on-a-chip platform based on microscale immiscible filtration to extract, concentrate and purify Neisseria gonorrhoeae DNA with an integrated detection assay based on colorimetric isothermal amplification. The platform was capable of detecting as low as 500 copies/mL from spiked synthetic urine and showed no cross-reactivity when challenged with DNAs from other common STIs. The credit card-size device allows DNA extraction and purification without power or centrifuges, and the detection reaction only needs a low-tech block heater, providing a straightforward and visual positive/negative result within 1 h. These advantages offer great potential for accurate, affordable and accessible monitoring of gonorrhea infection in resource-poor settings.

Genomic surveillance of SARS-COV-2 reveals diverse circulating variant lineages in Nairobi and Kiambu Counties, Kenya.

Genomic surveillance and identification of COVID-19 outbreaks are important in understanding the genetic diversity, phylogeny, and lineages of SARS-CoV-2. Genomic surveillance provides insights into circulating infections, and the robustness and design of vaccines and other infection control approaches. We sequenced 57 SARS-CoV-2 isolates from a Kenyan clinical population, of which 55 passed quality checks using the Ultrafast Sample placement on the Existing tRee (UShER) workflow. Phylo-genome-temporal analyses across two regions in Kenya (Nairobi and Kiambu County) revealed that B.1.1.7 (Alpha; n = 32, 56.1%) and B.1 (n = 9, 15.8%) were the predominant lineages, exhibiting low Ct values (5-31) suggesting high infectivity, and variant mutations across the two regions. Lineages B.1.617.2, B.1.1, A.23.1, A.2.5.1, B.1.596, A, and B.1.405 were also detected across sampling sites within target populations. The lineages and genetic isolates were traced back to China (A), Costa Rica (A.2.5.1), Europe (B.1, B.1.1, A.23.1), the USA (B.1.405, B.1.596), South Africa (B.1.617.2), and the United Kingdom (B.1.1.7), indicating multiple introduction events. This study represents one of the genomic SARS-CoV-2 epidemiology studies in the Nairobi metropolitan area, and describes the importance of continued surveillance for pandemic control.

Multi-repeat sequences identification using genome mining techniques for developing highly sensitive molecular diagnostic assay for the detection of Chlamydia trachomatis.

Chlamydia trachomatis ( C. trachomatis) is a common sexually transmitted infection (STI). In 2019, the World Health Organization reported about 131 million infections. The majority of infected patients are asymptomatic with cases remaining undetected. It is likely that missed C. trachomatis infections contribute to preventable adverse health outcomes in women and children. Consequently, there is an urgent need of developing efficient diagnostic methods. In this study, genome-mining approaches to identify identical multi-repeat sequences (IMRS) distributed throughout the C. trachomatis genome were used to design a primer pair that would target regions in the genome. Genomic DNA was 10-fold serially diluted (100pg/µL to 1×10 -3pg/µL) and used as DNA template for PCR reactions. The gold standard PCR using 16S rRNA primers was also run as a comparative test, and products were resolved on agarose gel. The novel assay, C. trachomatis IMRS-PCR, had an analytical sensitivity of 4.31 pg/µL, representing better sensitivity compared with 16S rRNA PCR (9.5 fg/µL). Our experimental data demonstrate the successful development of lateral flow and isothermal assays for detecting C. trachomatis DNA with potential use in field settings. There is a potential to implement this concept in miniaturized, isothermal, microfluidic platforms, and laboratory-on-a-chip diagnostic devices for reliable point-of-care testing.

Improving gonorrhoea molecular diagnostics: Genome mining-based identification of identical multi-repeat sequences (IMRS) in Neisseria gonorrhoeae.

Background: Curable sexually transmitted infections (STIs), such as Neisseria gonorrhoeae (N. gonorrhoeae), are a major cause of poor pregnancy outcomes. The infection is often asymptomatic in pregnant women, and a syndrome-based approach of testing leads to a missed diagnosis. Culture followed by microscopy is inadequate and time-consuming. The gold standard nucleic acid amplification tests require advanced infrastructure settings, whereas point-of-care tests are limited to immunoassays with sensitivities and specificities insufficient to accurately diagnose asymptomatic cases. This necessitates the development and validation of assays that are fit for purpose. Methods: We identified new diagnostic target biomarker regions for N. gonorrhoeae using an algorithm for genome mining of identical multi-repeat sequences (IMRS). These were then developed as DNA amplification primers to design better diagnostic assays. To test the primer pair, genomic DNA was 10-fold serially diluted (100 pg/µL to 1 × 10-3 pg/µL) and used as DNA template for PCR reactions. The gold standard PCR using 16S rRNA primers was also run as a comparative test, and both assay products were resolved on 1% agarose gel. Results: Our newly developed N. gonorrhoeae IMRS-PCR assay had an analytical sensitivity of 6 fg/µL representing better sensitivity than the 16S rRNA PCR assay with an analytical sensitivity of 4.3096 pg/µL. The assay was also successfully validated using clinical urethral swab samples. We further advanced this technique by developing an isothermal IMRS, which was both reliable and sensitive for detecting cultured N. gonorrhoeae isolates at a concentration of 38 ng/µL. Combining isothermal IMRS with a low-cost lateral flow assay, we were able to detect N. gonorrhoeae amplicons at a starting concentration of 100 pg/µL. Conclusion: Therefore, there is a potential to implement this concept within miniaturized, isothermal, microfluidic platforms, and laboratory-on-a-chip diagnostic devices for highly reliable point-of-care testing.

Antimicrobial sensitivity patterns of Staphylococcus species isolated from mobile phones and implications in the health sector.

OBJECTIVES: The aim of this research was to determine drug sensitivity profiles of Staphylococcus species isolated from mobile phones of students in Microbiology and Biomedical Laboratory Sciences from UZIMA University, Kisumu (Kenya) and the University Colleges Leuven-Limburg, Leuven (Belgium), respectively. RESULTS: All mobile phones (16/16, 100%) had gram-positive bacteria. 3/8 (37.5%) mobile devices had Staphylococcus aureus. 2/3 (67%) Staphylococcus aureus strains were resistant to ampicillin, oxacillin, ceftazidime, vancomycin and amoxicillin. Guidelines for disinfection of mobile phones need to be developed urgently to stop transmission of resistant bacteria.

Comparison of Microscopy and PCR for Detection of Giardia Lamblia and Entamoeba Histolytica in Human Stool Specimens in a Resource Limited Setting in Western Kenya.

BACKGROUND: Accurate diagnosis of Giardia lamblia and Entamoeba histolytica is important since these intestinal parasites account for a significant proportion of morbidity and mortality globally. Microscopy is the key diagnostic test used for diagnosis of the two parasites. Other tests including rapid diagnostic tests and polymerase chain reaction have been developed to improve the detection of these parasites. Most of these newer tests are not affordable in resource limited settings, hence the over reliance on microscopy. The objective of this study was to determine the reliability of microscopy in a resource limited setting in Western Kenya, a region endemic for the two intestinal parasites. METHODS: Polymerase chain reaction, the gold standard test, was performed on stool samples suspected for G. lamblia and E. histolytica. Microscopy was then performed on the same samples and the two tests compared. RESULTS: Microscopy was found to be 64.4% sensitive, 86.6% specific for the detection of G. lamblia. Additionally, this test was 64.2% sensitive and 83.6% specific for the diagnosis of E. histolytica. Cohen's kappa values of 0.51 and 0.47 were determined for microscopy for G. lamblia and E. histolytica respectively. McNemar's test revealed a significant difference between the two tests, P<0.001. CONCLUSION: This study found microscopy to be a reliable diagnostic test in this resource limited setting.

Novel biomarkers with promising benefits for diagnosis of cervical neoplasia: a systematic review.

BACKGROUND: Cervical cancer screening is slowly transitioning from Pappanicolaou cytologic screening to primary Visual Inspection with Acetic Acid (VIA) or HPV testing as an effort to enhance early detection and treatment. However, an effective triage tests needed to decide who among the VIA or HPV positive women should receive further diagnostic evaluation to avoid unnecessary colposcopy referrals is still lacking. Evidence from experimental studies have shown potential usefulness of Squamous Cell Carcinoma Antigen (SCC Ag), Macrophage Colony Stimulating Factor (M-CSF), Vascular Endothelial Growth Factor (VEGF), MicroRNA, p16INKa / ki-67, HPV E6/E7/mRNA, and DNA methylation biomarkers in detecting premalignant cervical neoplasia. Given the variation in performance, and scanty review studies in this field, this systematic review described the diagnostic performance of some selected assays to detect high-grade cervical intraepithelial neoplasia (CIN2+) with histology as gold standard. METHODS: We systematically searched articles published in English between 2012 and 2020 using key words from PubMed/Medline and SCOPUS with two reviewers assessing study eligibility, and risk of bias. We performed a descriptive presentation of the performance of each of the selected assays for the detection of CIN2 + . RESULTS: Out of 298 citations retrieved, 58 articles were included. Participants with cervical histology yielded CIN2+ proportion range of 13.7-88.4%. The diagnostic performance of the assays to detect CIN2+ was; 1) SCC-Ag: range sensitivity of 78.6-81.2%, specificity 74-100%. 2) M-CSF: sensitivity of 68-87.7%, specificity 64.7-94% 3) VEGF: sensitivity of 56-83.5%, specificity 74.6-96%. 4) MicroRNA: sensitivity of 52.9-67.3%, specificity 76.4-94.4%. 5) p16INKa / ki-67: sensitivity of 50-100%, specificity 39-90.4%. 6) HPV E6/E7/mRNA: sensitivity of 65-100%, specificity 42.7-90.2%, and 7) DNA methylation: sensitivity of 59.7-92.9%, specificity 67-98%. CONCLUSION: Overall, the reported test performance and the receiving operating characteristics curves implies that implementation of p16ink4a/ki-67 assay as a triage for HPV positive women to be used at one visit with subsequent cryotherapy treatment is feasible. For the rest of assays, more robust clinical translation studies with larger consecutive cohorts of women participants is recommended.

Distribution of Biomphalaria Snails in Associated Vegetations and Schistosome Infection Prevalence Along the Shores of Lake Victoria in Mbita, Kenya: A Cross-Sectional Study.

BACKGROUND: Schistosomiasis due to Schistosoma mansoni remains a major public health problem and cause of morbidity and mortality in sub-Saharan Africa despite the implementation of control programmes. More than 6 million Kenyans are at risk of infection. Regarding control measures, Biomphalaria snail species, which are the obligatory intermediate hosts for transmission of S. mansoni, have been neglected. Mbita subcounty in Homa Bay County, western Kenya, along Lake Victoria basin, has a high prevalence of S. mansoni infection despite mass drug administration. This study aimed to determine the abundance of Biomphalaria, with their associated vegetation and schistosome infection rates, along Mbita shoreline. METHODS: Sixteen purposively selected sites along the Mbita shoreline were sampled for Biomphalaria snails using a 30-minute scooping technique. Global positioning system technology was used to map selected sites. The associated vegetation at sampling sites were collected and identified. Schistosome infection status among the snails was determined via the detection of cercaria shedding. RESULTS: A total of 3,135 Biomphalaria sudanica snails were collected. The number of snails collected differed significantly between the 16 sites (F=11.735; degrees of freedom [df]=15.836; P<.001). Significant mean differences (MD) were also observed in terms of the number of snails collected per vegetation type (F=7.899; df=5.846; P<.001). The mean number of snails collected from Cyprus gracilis was significantly higher than that from Enydra fluactuants (MD= 2.03; P<.001), Eichhornia crassipes (MD=4.15; P<.010), and E. fluactuants mixed with E. crassipes (MD=2.516; P<.010). A total of 21 (0.67%) snails shed human cercariae, while 27 (0.86%) snails shed nonhuman cercariae, despite 14 sites having human faeces contamination. CONCLUSION: Although the schistosome infection prevalence among the snails was low, these sites may still be important exposure sites. C. gracilis is the main vegetation type associated with a high abundance of Biomphalaria snails. Molecular techniques are necessary for verification of schistosome positivity among the snails.

Seroprevalence and determinants of transfusion transmissible infections among voluntary blood donors in Homabay, Kisumu and Siaya counties in western Kenya.

OBJECTIVE: Since the implementation of a series of blood donation safety improvements in Kenya, information about seroprevalence and determinants of transfusion transmissible infections among voluntary blood donors especially in high HIV burden regions of Homabay, Kisumu and Siaya counties remain scanty. A cross-sectional study examining HIV, syphilis, hepatitis B and C virus sero-markers and associated determinants was conducted among voluntary blood donors. Their demographic characteristics and previous risk exposure were recorded in a pre-donation questionnaire, while blood samples collected were screened for hepatitis B, hepatitis C, human immunodeficiency viruses by ELISA and RPR (syphilis), then confirmed using CMIA. RESULTS: Overall TTIs seroprevalence was 114 (9.4%), distributed among HIV, HBV, HCV and syphilis at 14 (1.15%), 42 (3.46%), 39 (3.21%) and 19 (1.56%), respectively, with co-infections of 3 (0.25%). There were no significant differences in proportions distributions among demographic variables. However, high risk sex was significantly associated with higher odds of HBV infections [> 1 partner vs. 0-1 partner; odd ratio (OR) 2.60; 95% confidence interval (CI) 1.098-6.86; p = 0.046]. In conclusion, a substantial percentage of blood donors still harbor transfusion transmissible infections despite recent safety improvements with greater majority cases caused by HBV infections arising from previous exposure to high risk sex.

Correction to: Seroprevalence and determinants of transfusion transmissible infections among voluntary blood donors in Homabay, Kisumu and Siaya counties in western Kenya.

Following publication of the original article [1], the authors reported that for two of the authors, Felix Humwa and Vallarie Opollo, an incorrect affiliation has been given. In this Correction the incorrect and correct affiliations are listed.