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.

Detection of multidrug-resistant organisms of concern including Stenotrophomonas maltophilia and Burkholderia cepacia at a referral hospital in Kenya.

Regular monitoring of bacterial susceptibility to antibiotics in clinical settings is key for ascertaining the current trends as well as re-establish empirical therapy. This study aimed to determine bacterial contaminants and their antimicrobial susceptibility patterns from medical equipment, inanimate surfaces and clinical samples obtained from Thika Level V Hospital (TLVH), Thika, in Central Kenya. Three hundred and five samples were collected between the period of March 2021 to November 2021 and comprised urine, pus swabs, catheter swabs, stool, and environmental samples. Bacterial identification and antimicrobial susceptibility were performed using VITEK 2 and disc diffusion respectively. We observed that Coagulase-negative Staphylococci (28 /160, 17.5%) were the most commonly isolated species from clinical samples followed by E. coli (22 /160 13.8%) and S. aureus (22/160, 13.8%). The bed rails were the mostly contaminated surface with S. aureus accounting for 14.2% (6/42). Among the clinical samples, pus swabs yielded the highest number of pathogens was pus (92/160). Trauma patients had the highest proportion of isolates (67/160, 41.8%). High level of antimicrobial resistance to key antimicrobials, particularly among Enterobacterales was observed. Extended Spectrum Beta Lactamase (ESBL) phenotype was noted in 65.9% (29/44) of enteric isolates. While further ESBL genetic confirmatory studies are needed, this study highlights the urgent need for actions that mitigate the spread of antibiotic-resistant bacteria.

The impact of the COVID-19 pandemic on reproductive, maternal, neonatal, and child health care services in Kiambu County, Kenya.

The global response to COVID-19 undermined established public health goals. This study investigated the impact of COVID-19 on reproductive, maternal, neonatal, and child health (RMNCH) services in Kiambu County, Kenya. It was a retrospective cross-sectional study, where data on antenatal care (ANC), delivery, postnatal care (PNC), and family planning (FP) before and after COVID-19 was retrieved and compared. New ANC clients and 4th ANC visits decreased by 2.9% and 17% respectively. New clients attending PNC increased by 13.3% (p = 0.007). Skilled deliveries reduced by 0.3%, maternal, neonatal deaths, and fresh stillbirths reduced by 0.7%, 23.9%, and 15.8% respectively. Caesarean sections rose by 12.7% (p=0.001). New clients and revisits for family planning reduced by 15.4% and 6.6% respectively. The pandemic adversely affected most of the RMNCH services. There is a need for health departments to institute robust strategies to recover the gains lost during COVID-19.

La réponse mondiale à la COVID-19 a sapé les objectifs de santé publique établis. Cette étude a examiné l'impact du COVID-19 sur les services de santé reproductive, maternelle, néonatale et infantile (SRMNI) dans le comté de Kiambu, au Kenya. Il s'agissait d'une étude transversale rétrospective, dans laquelle les données sur les soins prénatals (ANC), l'accouchement, les soins postnatals (PNC) et la planification familiale (PF) avant et après la COVID-19 ont été récupérées et comparées. Les nouvelles clientes de CPN et les 4èmes visites de CPN ont diminué respectivement de 2,9 % et 17 %. Les nouveaux clients fréquentant la PNC ont augmenté de 13,3 % (p = 0,007). Les accouchements qualifiés ont diminué de 0,3 %, les décès maternels et néonatals et les nouvelles mortinaissances ont diminué respectivement de 0,7 %, 23,9 % et 15,8 %. Les césariennes ont augmenté de 12,7 % (p=0,001). Les nouveaux clients et les nouvelles visites pour la planification familiale ont diminué respectivement de 15,4% et 6,6%. La pandémie a eu des conséquences néfastes sur la plupart des services de RMNCH. Il est nécessaire que les services de santé mettent en place des stratégies solides pour récupérer les gains perdus pendant la COVID-19.

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.

Application of Hybridization Chain Reaction/CRISPR-Cas12a for the Detection of SARS-CoV-2 Infection.

Globally, the emergence of the coronavirus disease (COVID-19) has had a significant impact on life. The need for ongoing SARS-CoV-2 screening employing inexpensive and quick diagnostic approaches is undeniable, given the ongoing pandemic and variations in vaccine administration in resource-constrained regions. This study presents results as proof of concept to use hybridization chain reaction (HCR) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a complex for detecting SARS-CoV-2. HCR hairpin probes were designed using the NUPACK web-based program and further used to amplify the SARS-CoV-2 N gene in archived nasopharyngeal samples. The results were visualized using agarose gels and CRISPR Cas12a-based lateral flow strips. The assay was evaluated using the gold standard, real-time polymerase chain reaction (RT-PCR), as recommended by the World Health Organization (WHO). The results show the comparative efficiency of HCR to RT-PCR. This study shows that HCR and CRISPR are viable alternatives for diagnosing SARS-CoV-2 in samples.

Adaptive immune receptor features related to breast cancer tissue in Kenyan patients: high immunoglobulin gene expression and high levels of gamma-delta T-cells.

BACKGROUND: Characterization of the breast cancer (BC) immune response may provide information for a point of intervention, such as application of immunotherapeutic treatments. In this study, we sought to recover and characterize the adaptive immune receptor (IR) recombination reads from genomics files representing Kenyan patients, to better understand the immune response specifically related to those patients. METHODS: We used a previously applied algorithm and software to obtain productive IR recombination reads from cancer and adjacent normal tissue samples representing 22 Kenyan BC patients. RESULTS: From both the RNAseq and exome files, there were significantly more T-cell receptor (TCR) recombination reads recovered from tumor samples compared to marginal tissue samples. Also, the immunoglobulin (IG) genes were expressed at a much higher level than the TCR genes (p-value = 0.0183) in the tumor samples. And, the tumor IG CDR3s consistently represented more positively charged amino acid R-groups, in comparison to the marginal tissue, IG CDR3s. CONCLUSION: For Kenyan patients, a high level of IG expression, representing specific CDR3 chemistries, was associated with BC. These results lay the foundation for studies that could support specific immunotherapeutic interventions for Kenyan BC patients.

Cancer referrals at African Inland Church Kijabe Hospital, Kiambu County (2014-2020) and the impact of COVID-19.

In Kenya, cancer is the third leading cause of death. The African Inland Church Kijabe Hospital (AICKH) is a level 4 missionary hospital. The hospital serves the Kenyan population in many areas, including cancer care, and some of these services were affected during the coronavirus disease 2019 (COVID-19) pandemic. We aimed to leverage a recently established hospital-based cancer registry of patients treated at AICKH between 2014 and 2020 to describe the cancer cases and patient referral patterns seen at AICKH during the COVID-19 pandemic in 2020. A cross-sectional retrospective survey was conducted through medical records abstraction in the surgery, breast clinic, palliative care and pathology departments. A total of 3279 cases were included in the study, with females accounting for 58.1% of the cases. The top-three cancers overall were breast (23.0%), oesophagus (20.5%) and prostate (8.6%). There was a minimal increase in the number of cancer cases in 2020 (1.7%) compared with 2019, with an increase of 19.3% in 2019 compared with 2018. In conclusion, AICKH is one of the few hospitals in Kenya where a large number of cancer patients seek healthcare, and referral of cancer cases changed in 2020, which may be due to the COVID-19 pandemic. Future efforts can leverage this registry to determine the impacts of cancer diagnosis and treatment on survival outcomes.

A sample-to-answer COVID-19 diagnostic device based on immiscible filtration and CRISPR-Cas12a-assisted detection.

In response to the ongoing coronavirus disease 2019 (COVID-19) pandemic and disparities of vaccination coverage in low-and middle-income countries, it is vital to adopt a widespread testing and screening programme, combined with contact tracing, to monitor and effectively control the infection dispersion in areas where medical resources are limited. This work presents a lab-on-a-chip device, namely 'IFAST-LAMP-CRISPR', as an affordable, rapid and high-precision molecular diagnostic means for detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The herein proposed 'sample-to-answer' platform integrates RNA extraction, amplification and molecular detection with lateral flow readout in one device. The microscale dimensions of the device containing immiscible liquids, coupled with the use of silica paramagnetic beads and guanidine hydrochloride, streamline sample preparation (including RNA extraction, concentration and purification) in 15 min with minimal hands-on steps. The pre-amplification in combination with CRISPR-Cas12a detection assays targeting the nucleoprotein (N) gene achieved visual identification of ≥ 470 copies mL-1 genomic SARS-CoV-2 samples in 45 min. On-chip assays showed the ability to isolate and detect SARS-CoV-2 RNA from 100 genome copies mL-1 of replication-deficient viral particles in 1 h. This simple, affordable and integrated platform demonstrated a visual, faster, and yet specificity- and sensitivity-comparable alternative to the costly gold-standard reverse transcription-polymerase chain reaction (RT-PCR) assay, requiring only a simple heating source. Initial testing illustrates the platform viability both on nasopharyngeal swab and saliva samples collected using the easily accessible Swan-brand cigarette filter, providing a complete workflow for COVID-19 diagnostics in low-resource settings.

The impact of measures to curb COVID-19 on patient attendance at 10 hospitals in Machakos County, Kenya.

BACKGROUND: The COVID-19 pandemic has resulted in both direct and indirect impacts on patients and population health. To better understand the impact of the measures put in place by the Kenyan government on health care provision, this project sought to document and quantify the impact of the restriction measures on patients' attendance in Machakos County. METHODS: Hospital attendance at 10 public hospitals were obtained including Machakos Level 5 Hospital (the county referral facility) and one health facility from each of the 9 Sub-counties of Machakos County. Data on outpatient and inpatient attendance from November 2019 to May 2020 were obtained and compared with a similar calendar period from the previous year. Key informant interviews (KIIs), focused group discussions (FGDs) and in-depth interviews were conducted with the hospital management personnel (n = 46), as well as with the patients (n = 453) who missed scheduled clinic appointments at Machakos Level 5 Hospital to understand the reasons behind the drop in attendance numbers. RESULTS: Overall, there was a decline in the number of patient attendances compared to the prior calendar period. Outpatient attendance reduced by 24.7% in April 2020 (n = 39 704) compared with April 2019 (n = 52 731). Inpatient attendance reduced by 13.7% in April 2020 (n = 3298) compared with April 2019 (n = 2845). Declines in patient attendance were observed in all hospitals that had inpatient services. A great decline in attendance was noted among larger hospitals that run specialty clinics, which were suspended mid-March 2020 when the first case of COVID-19 was announced. Some increase in attendance was noted in May when most clinics resumed operations. Most hospital management staff highlighted the closure of clinics as the main reason for reduced attendance while patients added that they also feared contracting COVID-19 at the hospital and the stigma they would face should they be quarantined. CONCLUSIONS: The findings from this study provide evidence that the COVID-19 pandemic outbreak and measures put in place by the government to curb its spread disrupted the provision of health services in Machakos County. Efforts to minimize adverse impacts of indirect impacts on access to health care and preventative services to counter increased morbidity and mortality require attention throughout the pandemic.

Development and Clinical Validation of Iso-IMRS: A Novel Diagnostic Assay for P. falciparum Malaria.

In many countries targeting malaria elimination, persistent malaria infections can have parasite loads significantly below the lower limit of detection (LLOD) of standard diagnostic techniques, making them difficult to identify and treat. The most sensitive diagnostic methods involve amplification and detection of Plasmodium DNA by polymerase chain reaction (PCR), which requires expensive thermal cycling equipment and is difficult to deploy in resource-limited settings. Isothermal DNA amplification assays have been developed, but they require complex primer design, resulting in high nonspecific amplification, and show a decrease in sensitivity than PCR methods. Here, we have used a computational approach to design a novel isothermal amplification assay with a simple primer design to amplify P. falciparum DNA with analytical sensitivity comparable to PCR. We have identified short DNA sequences repeated throughout the parasite genome to be used as primers for DNA amplification and demonstrated that these primers can be used, without modification, to isothermally amplify P. falciparum parasite DNA via strand displacement amplification. Our novel assay shows a LLOD of ∼1 parasite/µL within a 30 min amplification time. The assay was demonstrated with clinical samples using patient blood and saliva. We further characterized the assay using direct amplicon next-generation sequencing and modified the assay to work with a visual readout. The technique developed here achieves similar analytical sensitivity to current gold standard PCR assays requiring a fraction of time and resources for PCR. This highly sensitive isothermal assay can be more easily adapted to field settings, making it a potentially useful tool for malaria elimination.

Leveraging technology for health services continuity in times of COVID-19 pandemic: Patient follow-up, and mitigation of worse patient outcomes.

BACKGROUND: Since the outbreak of the COVID-19 pandemic in Wuhan, China, which has now spread globally, the health systems continue to face challenges in the provision of health care, there is a risk of exposure for both the physicians and the patients. While there is significant progress in the adoption of technology in health care. This study sought to examine the adverse effects of the measures put in place by the government to curb the spread of COVID-19 and come up with an intervention to prevent worse outcomes for chronic conditions. METHODS: Booking registers for four specialty clinics in Machakos Level 5 Hospital were reviewed to identify patients who missed clinic appointments for follow-up. An automated data collection tool (ODK-collect) was used for data collection. COVID-19 Machakos App was developed to facilitate follow-up and referral of patients to the nearest facilities, capturing and posting of information in real-time to a central database. The mobile App also facilitated the tracking of patients and aided doctors to give feedback on whether the patients reported to the referred facilities. The doctors were also able to capture doctors' notes on the patients' status while ensuring the confidentiality and privacy of the patients. An interactive dashboard was developed to generate analytics reports and summaries to monitor clinic attendance and trends in the provision of health care during the pandemic period. RESULTS: Register data showed 977 (81.5%) out of a total of 1199 patients had missed their scheduled appointments. Among the 977, 746 (76%) were residents of Machakos County and qualified for follow-up. Missed appointments varied by clinic: Cancer Clinic 12 (1.6) %), Diabetes Clinic 212 (28.4%), Hypertension 293 (39.3%), and Paediatrics Clinic 229 (30.7%). Contact was made and follow-up was attempted for 746 patients, of which 453 patients (60.7%) were successful. The follow-up distribution of the 453 patients varied by the clinic as follows: Cancer Clinic 10, Diabetes Clinic 146, Hypertension 185, and Paediatrics Clinic 112. During the follow-up process, 331 patients from diabetes and hypertension clinics were requested to choose a preferred or nearby facility to be referred to. 191 (58%) patients chose Machakos Level 5 Hospital as their preferred facility and 137 (41%) patients chose to be referred to level 3 or 4 hospitals within the County. Three deaths were reported from the medical (Hypertension) clinic. Through the developed App, a total, 82 (60%) patients out of the 137 were reviewed at the referral facilities jointly with a specialist at Machakos Level 5 Hospital. For the duration of the study, some patients reported worse conditions by the time of review after missing scheduled appointments. CONCLUSIONS: This intervention demonstrated that mobile phone technology could be leveraged to provide specialty treatment services remotely to mitigate against worse patient outcomes. The study reveals that there is a dire need to integrate technological interventions in the provision of health care services to ensure continuity.

Exploring perspectives on antimicrobial stewardship: a qualitative study of health managers in Kenya.

BACKGROUND: Antimicrobial resistance is a significant public health concern with the establishment of antimicrobial stewardship in hospitals being increasingly obligatory. Perspectives and insights of health managers on antimicrobial stewardship (AMS), complementary health services and building blocks are imperative towards implementation of robust AMS programs. This study aimed to understand perspectives of hospital managers on AMS and identify areas of management engagement while addressing potential blockades to change. METHODS: A cross-sectional, qualitative, multicenter study was conducted in three hospitals in Kenya. Key-informant interviews on perspectives on AMS were administered to hospital managers. Qualitative data was captured using audio tapes and field notes, transcribed and managed using NVivo 12 software. An iterative process was used to develop the thematic framework and updated in two rounds of iteration analysis. Analysis charts for each emergent theme were developed and categorized across all participants. RESULTS: Perspectives on AMS are described in five thematic categories; Importance of antimicrobial stewardship and the role of medicines and therapeutics committee, availability of antimicrobial formulary and usage surveillance systems, laboratory competency and recommendations for infection prevention and management, educational resources and communications channels available, building blocks and low-lying fruits for Antimicrobial Stewardship Committees. The role of stewardship collaboration in diagnosis and antimicrobial prescription was alluded to with managers indicating a growing rise in occurrence of antimicrobial resistance. There lacked contextualized, hospital specific antimicrobial formulary and adequate laboratory competency. Staff training and communication channels were available in varying capacity across the three hospitals. Building blocks identified include medicines and therapeutics committee, education, and training platforms (Continuous Medical Education and Continuous Professional Development activities) and hospital leadership commitment. CONCLUSIONS: The practice of antimicrobial stewardship is not implemented and well developed as demonstrated by lack of core AMS complementary health services. However, the health managers are aware of the fundamental importance of antimicrobial stewardship programs and the vast benefits of implementation and institutionalization of AMS to hospitals and their clients. The findings underpin the importance of understanding and incorporating perspectives of health managers on existing contextual mechanisms that can be leveraged on to establish robust AMS programs in the fight against antimicrobial resistance.

Combating antibiotic resistance using guidelines and enhanced stewardship in Kenya: a protocol for an implementation science approach.

INTRODUCTION: Antimicrobial resistance (AMR) is a growing problem globally especially in Sub-Saharan Africa including Kenya. Without any intervention, lower/middle-income countries (LMICs) will be most affected due to already higher AMR levels compared with higher income countries and due to the far higher burden of diseases in the LMICs. Studies have consistently shown that inappropriate use of antimicrobials is the major driver of AMR. To address this challenge, hospitals are now implementing antibiotic stewardship programmes (ASPs), which have been shown to achieve reduced antibiotic usage, to decrease the prevalence of resistance and lead to significant economic benefits. However, the implementation of the guideline is highly dependent on the settings in which they are rolled out. This study, employing an implementation science approach, aims to address the knowledge gap in this area and provide critical data as well as practical experiences when using antibiotic guidelines and stewardship programmes in the public health sector. This will provide evidence of ASP performance and potentially contribute to the county, national and regional policies on antibiotics use. METHODS AND ANALYSIS: The study will be conducted in three geographically diverse regions, each represented by two hospitals. A baseline study on antibiotic usage, resistance and de-escalation, duration of hospital stay, rates of readmission and costs will be carried out in the preimplementation phase. The intervention, that is, the use of antibiotic guidelines and ASPs will be instituted for 18 months using a stepwise implementation strategy that will facilitate learning and continuous improvement of stewardship activities and updating of guidelines to reflect the evolving antibiotic needs. ETHICS AND DISSEMINATION: Approvals to carry out the study have been obtained from the National Commission for Science, Technology and Innovation and the Mount Kenya University Ethics Review Committee. The approvals from the two institutions were used to obtain permission to conduct the study at each of the participating hospitals. Study findings will be presented to policy stakeholders and published in peer-reviewed scientific journals. It is anticipated that the findings will inform the appropriate antibiotic use guidelines within our local context.