Evaluation of lethality and cytotoxic effects induced by Naja ashei (large brown spitting cobra) venom and the envenomation-neutralizing efficacy of selected commercial antivenoms in Kenya.

Neutralization of lethality in mice model at the preclinical level has been established by the World Health Organization as the gold standard for the evaluation of antivenom efficacy. The assessment of the neutralization profiles of antivenoms helps to discern the efficacy or otherwise of these antivenoms at neutralizing the toxic effects induced by medically significant snake venoms. However, for many antivenoms, information on their preclinical efficacy remains limited. Therefore, to strengthen global efforts at reducing the impact of snakebite envenoming, the provision of information on the preclinical efficacy of antivenoms, especially in parts of the world where antivenom availability and accessibility is problematic, including sub-Saharan Africa is crucial. This study presents the lethal and toxic activities of N. ashei venom and the neutralizing capacity of two commonly used commercial antivenoms in Kenya; VINS™ and Inoserp™. Median lethal dose (LD50), minimum necrotizing dose (MND) and minimum edema-forming dose (MED) of N. ashei venom as well as the neutralization of these effects were evaluated in mice. The LD50 of N. ashei venom was found to be 4.67 (3.34-6.54) mg/kg while MND and MED were 11.00 µg and 0.80 µg respectively. Both VINS™ and Inoserp™ antivenoms demonstrated capacity to neutralize the lethal and toxic effects induced by Naja ashei venom albeit at varying efficacies. Our results thus confirm the toxic effects of N. ashei venom as previously observed with other Naja sp. venoms and also underscore the relevance of para-specific neutralizing capacity of antivenoms in the design of antivenoms.

Development and Characterization of Anti-Naja ashei Three-Finger Toxins (3FTxs)-Specific Monoclonal Antibodies and Evaluation of Their In Vitro Inhibition Activity.

Antivenom immunotherapy is the mainstay of treatment for snakebite envenoming. Most parts of the world affected by snakebite envenoming depend on broad-spectrum polyspecific antivenoms that are known to contain a low content of case-specific efficacious immunoglobulins. Thus, advances in toxin-specific antibodies production hold much promise in future therapeutic strategies of snakebite envenoming. We report anti-3FTxs monoclonal antibodies developed against N. ashei venom in mice. All the three test mAbs (P4G6a, P6D9a, and P6D9b) were found to be IgG antibodies, isotyped as IgG1. SDS-PAGE analysis of the test mAbs showed two major bands at approximately 55 and 29 kDa, suggestive of immunoglobulin heavy and light chain composition, respectively. The immunoaffinity-purified test mAbs demonstrated higher binding efficacy to the target antigen compared to negative control. Similarly, a cocktail of the test mAbs was found to induce a significantly higher inhibition (p-value < 0.0001) compared to two leading commercial brands of antivenoms on the Kenyan market, implying a higher specificity for the target antigen. Both the test mAbs and 3FTxs polyclonal antibodies induced comparable inhibition (p-value = 0.9029). The inhibition induced by the 3FTxs polyclonal antibodies was significantly different from the two antivenoms (p-value < 0.0001). Our results demonstrate the prospects of developing toxin-specific monoclonal-based antivenoms for snakebite immunotherapy.

In vitro anticoccidial activity of nanoencapsulated bromelain against Eimeria spp. oocysts isolated from goats in Kenya.

Background and Aim: The emergence of drug-resistant strains of Eimeria spp. calls for the development of novel anticoccidial drugs. Plant extracts provide a possible natural source for such drugs. This study aimed to investigate the in vitro anticoccidial activity of encapsulated bromelain (EB) in chitosan nanocarriers on Eimeria spp. oocysts isolated from goats kept by farmers in Kenya. Materials and Methods: Bromelain was extracted from the peel of ripe pineapples using standard methods. Eimeria spp. oocysts were isolated from the feces of goats using a flotation method. The inhibition of sporulation was assayed after exposing the oocysts to solutions of EB, non-EB (NEB), and diclazuril (positive control) at concentrations between 4 mg/mL and 0.125 mg/mL for 48 h. The oocysts were examined under a microscope (40x) to determine the effects of the drugs on the sporulation process. The percentage of sporulation inhibition was calculated after 48 h and the inhibition concentration 50% (IC50) was determined by probit analysis. Results: Bromelain manifested anticoccidial activity through the inhibition of the sporulation of coccidia oocysts. EB achieved inhibition with a lower dose compared with NEB. The IC50 values of diclazuril, EB, and NEB were 0.078 mg/mL, 0.225 mg/mL, and 0.575 mg/mL, respectively. There were significant differences (p<0.01) between the IC50 of EB and NEB compared with the standard treatment drug. Conclusion: This preliminary study showed that EB has anticoccidial activity supporting further evaluation at an in vivo level to develop a novel drug for the management of coccidiosis in goats.

Development of an Inhibition Enzyme-Linked Immunosorbent Assay (ELISA) Prototype for Detecting Cytotoxic Three-Finger Toxins (3FTxs) in African Spitting Cobra Venoms.

The administration of toxin-specific therapy in snake envenoming is predicated on improved diagnostic techniques capable of detecting specific venom toxins. Various serological tests have been used in detecting snakebite envenoming. Comparatively, enzyme-linked immunosorbent assay (ELISA) has been shown to offer a wider practical application. We report an inhibition ELISA for detecting three-finger toxin (3FTx) proteins in venoms of African spitting cobras. The optimized assay detected 3FTxs in N. ashei (including other Naja sp.) venoms, spiked samples, and venom-challenged mice samples. In venoms of Naja sp., the assay showed inhibition, implying the detection of 3FTxs, but showed little or no inhibition in non-Naja sp. In mice-spiked samples, one-way ANOVA results showed that the observed inhibition was not statistically significant between spiked samples and negative control (p-value = 0.164). Similarly, the observed differences in inhibition between venom-challenged and negative control samples were not statistically significant (p-value = 0.9109). At an LOD of 0.01 µg/mL, the assay was able to confirm the presence of 3FTxs in the samples. Our results show a proof of concept for the use of an inhibition ELISA model as a tool for detecting 3FTxs in the venoms of African spitting cobra snakes.

Increase in the Immune Response in Balb/c Mice after the Co-Administration of a Vector-Based COVID-19 Vaccine with Cytosine Phosphoguanine Oligodeoxynucleotide.

The effects of cytosine phosphoguanine oligodeoxynucleotides (CPG ODNs) on immune response have been demonstrated for different vaccines; however, such information is limited for the vector-based Coronavirus disease 2019 (COVID-19). This paper aims to demonstrate the potential effect of CPG ODNs on immunological response against the vector-based COVID-19 vaccine on Balb/c mice using a JNJ-78436735 Ad26.COV2-S recombinant as a model vaccine. A total of 18 BALB/c mice clustered into six groups were used. All groups were observed for 14- and 28-days post immunization. Qualitative determination of IgG was performed using indirect Enzyme-Linked Immunosorbent Assay (ELISA) and qPCR for cytokine profiling. A significant (p ≤ 0.001) rise in antibody response was observed for groups 3 and 4, who also showed increased expression levels of Tumor Necrosis Factor (TNF) and Interferon Gamma (IFN-γ). Immunological parameters for toxicity were normal in all treatment groups. We conclude that supplementing vector-based COVID-19 vaccines with CpG ODNs has the potential to boost the body's immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Preliminary In Vivo Evidence of Oral Selenium Supplementation as a Potentiating Agent on a Vector-Based COVID-19 Vaccine in BALB/c Mice.

Evidence of efficacy and toxicity of oral selenium supplementation in vaccine administration against severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) in mice models is scarce. In this study, 4 × 109 virus particles (40 µL) dose of Janssen COVID-19 intramuscular injection vaccine was supplemented with a commercial selenium supplement and sodium selenite orally in BALB/c mice (N = 18). Qualitative determination of anti-spike IgG antibody response using indirect Enzyme-Linked Immunosorbent Assay (ELISA) showed significant (p ≤ 0.001) increase in anti-spike IgG antibody response for mice groups immunized with vaccine and supplemented selenium. Furthermore, cytokine profiling using real-time quantitative polymerase chain reaction also showed an increase in IL-6 and IL-10 mRNA levels normalized using hypoxanthine phosphoribosyl transferase 1 (Hprt1) and glyceraldehyde 3-phosphate dehydrogenase (Gadph) housekeeping genes. There was no statistical significance (p < 0.465) among treated and untreated groups for alanine transaminase (ALT), aspartate transaminase (AST), urea, and creatinine parameters. The study presents preliminary findings and suggests that supplementing Janssen COVID-19 vaccines with selenium can generate more robust immune responses.

Pre-evaluation assessment of serological-based COVID-19 point-of-care lateral flow assays in Kenya.

BACKGROUND: Timely testing is a key determinant of management outcomes of coronavirus disease 2019 (COVID-19). Real-time reverse transcription polymerase chain reaction tests are currently the mainstay for COVID-19 testing. However, serological point-of-care tests (PoCTs) can be useful in identifying asymptomatic and recovered cases, as well as herd immunity. OBJECTIVE: The aim of this study was to assess COVID-19 PoCTs in Kenya to support the emergency use authorisation of these tests. METHODS: Between March 2020 and May 2020, 18 firms, of which 13 were from China, submitted their PoCTs to the national regulatory authority, the Pharmacy and Poison Board, who in turn forwarded them to the Kenya Medical Research Institute for pre-evaluation assessment. The tests were run with real-time reverse transcription polymerase chain reaction COVID-19-positive samples. Pre-COVID-19 plasma samples that were collected in June 2019 were used as negative samples. The shelf lives of the PoCTs ranged from 6 to 24 months. RESULTS: Only nine (50%) tests had sensitivities ≥ 40% (range: 40% - 60%) and the ability of these tests to detect IgM ranged from 0% to 50%. Many (7/18; 38.9%) of the kits had very weak IgM and IgG band intensities (range: 2-3). CONCLUSION: Serological-based PoCTs available in Kenya can only detect COVID-19 in up to 60% of the infected population.

Carica papaya Leaf Extract Silver Synthesized Nanoparticles Inhibit Dengue Type 2 Viral Replication In Vitro.

The current global occurrence of dengue infection annually is approximately 400 million, with a case fatality rate of 2.5%. However, there are no antiviral agents. Carica papaya leaf extract is known for its medicinal value, due to the presence of organic compounds that possess antimicrobial, anti-inflammatory, and antioxidant activities. This study determined the anti-dengue effect of C. papaya leaf extract silver synthesized nanoparticles. In this study, aqueous and non-aqueous extractions were carried out, followed by the synthesis of silver nanoparticles as well as characterization through Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy. The in vitro anti-dengue effect was evaluated using a focus reduction neutralization test on kidney Vero E2 cell lines. In silico studies involved molecular docking to determine the potential interactions between the bioactive compounds in C. papaya leaf extract and the viral NS5 protein. C. papaya leaf methanol extract silver synthesized nanoparticle was the most promising with an IC50 of 9.20 µg/mL. Molecular docking showed 5,7 dimethoxycoumarin as the best ligand, with binding energy of -7.75 kcal/mol, indicating high affinity for the NS5 protein. These results highlight that C. papaya leaf methanol extract silver synthesized nanoparticles could be used to inhibit dengue virus type 2 viral replication. However, we recommend further studies to determine their toxicity and the safety profiles.

Pre-evaluation assessment of serological-based COVID-19 point-of-care lateral flow assays in Kenya

Background: Timely testing is a key determinant of management outcomes of coronavirus disease 2019 (COVID-19). Real-time reverse transcription polymerase chain reaction tests are currently the mainstay for COVID-19 testing. However, serological point-of-care tests (PoCTs) can be useful in identifying asymptomatic and recovered cases, as well as herd immunity. Objective: The aim of this study was to assess COVID-19 PoCTs in Kenya to support the emergency use authorisation of these tests. Methods: Between March 2020 and May 2020, 18 firms, of which 13 were from China, submitted their PoCTs to the national regulatory authority, the Pharmacy and Poison Board, who in turn forwarded them to the Kenya Medical Research Institute for pre-evaluation assessment. The tests were run with real-time reverse transcription polymerase chain reaction COVID-19-positive samples. Pre-COVID-19 plasma samples that were collected in June 2019were used as negative samples. The shelf lives of the PoCTs ranged from 6 to 24 months. Results: Only nine (50%) tests had sensitivities ≥ 40% (range: 40% ­ 60%) and the ability of these tests to detect IgM ranged from 0% to 50%. Many (7/18; 38.9%) of the kits had very weak IgM and IgG band intensities (range: 2­3). Conclusion: Serological-based PoCTs available in Kenya can only detect COVID-19 in up to 60% of the infected population.

Evaluation of a program to improve hand hygiene in Kenyan hospitals through production and promotion of alcohol-based Handrub - 2012-2014.

Although critical to prevent healthcare-associated infections, hand hygiene (HH) compliance is poor in resource-limited settings. In 2012, three Kenyan hospitals began onsite production of alcohol-based handrub (ABHR) and HH promotion. Our aim is to determine the impact of local production of ABHR on HH compliance and perceptions of ABHR. We observed 25,738 HH compliance opportunities and conducted 15 baseline and post-intervention focus group discussions. Hand Hygiene compliance increased from 28% (baseline) to 38% (post-intervention, p = 0.0003). Healthcare workers liked the increased accessibility of ABHR, but disliked its smell, feel, and sporadic availability. Onsite production and promotion of ABHR resulted in modest HH improvement. Enhancing the quality of ABHR and addressing logistical barriers could improve program impact.

Unclear association between levels of Plasmodium falciparum lactate dehydrogenase (PfLDH) in saliva of malaria patients and blood parasitaemia: diagnostic implications?

BACKGROUND: The use of saliva in diagnosis of infectious diseases is an attractive alternative to procedures that involve blood drawing. It promises to reduce risks associated with accidental needle pricks and improve patient compliance particularly in malaria survey and drug efficacy studies. Quantification of parasitaemia is useful in establishing severity of disease and in assessing individual patient response to treatment. In current practice, microscopy is the recommended technique, despite its limitations. This study measured the levels of Plasmodium falciparum lactate dehydrogenase (PfLDH) in saliva of malaria patients and investigated the relationship with blood parasitaemia. METHODS: Matched pre-treatment blood and saliva samples were collected from patients at Msambweni District Hospital, Kenya. Parasitaemia was determined and only those confirmed to be Plasmodium falciparum mono-infected were recruited. PfLDH was quantified in saliva using a commercial ELISA kit. A total of 175 samples were collected. Relationship between blood parasitaemia and concentration of PfLDH in saliva was determined using Pearson correlation statistics. F test was used to determine whether there is a significant difference between levels of PfLDH in saliva of patients with moderate to high parasitaemia and those with low parasitaemia. RESULTS: One-hundred and seventy-five patient samples were positive for malaria by microscopy. Of these, 62 (35%) tested positive for PfLDH in saliva, 113 (65%) were false negatives. For those that tested positive, (53) 85% were from patients with moderate to high parasitaemia while 9 (15%) were from patients with low parasitaemia. A correlation co-efficient of 0.18 indicated a weak positive relationship between the concentration of PfLDH in saliva and blood parasitaemia. There was a marginal difference between levels of PfLDH in saliva of patients with moderate to high parasitaemia and those with low parasitaemia [F (1, 59) = 1.83, p = 0.1807]. CONCLUSION: The results indicate that there is a weak correlation between levels of PfLDH in saliva and blood parasitaemia. This is weak association could be as a result of low sensitivity of the assay used as well as presence of inhibitors and proteases in saliva. Further studies should be focused towards reducing the number of false negatives and developing a customised assay that is specific for detection of PfLDH in saliva.

Anti-bacterial efficacy of alcoholic hand rubs in the Kenyan market, 2015.

BACKGROUND: Hand hygiene is known to be effective in preventing hospital and community-acquired infections. The increasing number of hand sanitizer brands in Kenyan hospitals and consumer outlets is of concern. Thus the main aim of this study was to evaluate the anti-bacterial efficacy and organoleptic properties of these hand sanitizers in Kenya. METHODS: This was an experimental, laboratory-based study of 14 different brands of hand sanitizers (coded HS1-14) available in various retail outlets and hospitals in Kenya. Efficacy was evaluated using standard non-pathogenic Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATCC 27853) as per the European Standard (EN). The logarithmic reduction factors (RF) were assessed at baseline and after treatment, and log reduction then calculated. Ten and 25 healthy volunteers participated in the efficacy and organoleptic studies respectively. RESULTS: Four (28.6%) hand sanitizers (HS12, HS9, HS13 and HS14) showed a 5.9 reduction factor on all the three bacteria strains. Seven (50%) hand sanitizers had efficacies of <3 against all the three bacteria strains used. Efficacy on E. Coli was higher compared to the other pathogens. Three hand sanitizers were efficacious on one of the pathogens and not the other. In terms of organoleptic properties, gel-based formulations were rated far higher than the liquid based formulations brands. CONCLUSION: Fifty percent (50%) of the selected hand sanitizers in the Kenyan market have efficacy that falls below the World Health Organization (WHO) and DIN EN 1500:2013. Of the 14 hand sanitizers found in the Kenyan market, only four showed efficacies that were comparable to the WHO-formulation. There is a need to evaluate how many of these products with <3 efficacy that have been incorporated into the health system for hand hygiene and the country's policy on regulations on their usage.

Hepatitis B infection is highly prevalent among patients presenting with jaundice in Kenya.

BACKGROUND: Viral hepatitis is a major concern worldwide, with hepatitis A (HAV) and E (HEV) viruses showing sporadic outbreaks while hepatitis B (HBV) and C (HCV) viruses are associated with chronic hepatitis, cirrhosis and hepatocellular carcinoma. The present study determined the proportion, geographic distribution and molecular characterization of hepatitis viruses among patients seeking medical services at hospitals throughout Kenya. METHODS: Patients presenting with jaundice at four selected hospitals were recruited (n = 389). Sera were tested for the presence of antibody to hepatitis viruses A through E, and HBV surface antigen (HBsAg). Nucleic acid from anti-HAV IgM antibody and HBsAg positive samples was extracted, amplified and sequenced. RESULTS: Chronic HBV infection was the leading cause of morbidity among patients with symptoms of liver disease seeking medical help. Incident HCV, HEV and HDV infection were not detected among the patients in this study, while the proportion of acute HAV was low; HAV IgM positivity was observed in 6.3 % of patients and sequencing revealed that all cases belonged to genotype 1B. HCV seropositivity upon initial screening was 3.9 % but none were confirmed positive by a supplementary immunoblot assay. There was no serological evidence of HDV and acute HEV infection (anti-HEV IgM). HBsAg was found in 50.6 % of the patients and 2.3 % were positive for IgM antibody to the core protein, indicating probable acute infection. HBV genotype A was predominant (90.3 %) followed by D (9.7 %) among HBV DNA positive specimens. Full genome analysis showed HBV/D isolates having similarity to both D4 and D6 subgenotypes and D/E recombinant reference sequences. Two recombinant sequences demonstrated > 4 % nucleotide divergence from other previously known D/E recombinants. CONCLUSIONS: HBV is highly prevalent among patients seeking care for symptoms consistent with hepatitis, compared to the general population. Molecular characterization of HBV isolates indicated recombinant strains that may give rise to new circulating variants. There is a need to document the prevalence, clinical manifestation and distribution of the variants observed. HAV genotype 1B, prevalent in Africa, was observed; however, the absence of HCV, HDV and acute HEV in this study does not rule out their presence in Kenya.

Prevalence, awareness and risk factors associated with Hepatitis B infection among pregnant women attending the antenatal clinic at Mbagathi District Hospital in Nairobi, Kenya.

INTRODUCTION: Hepatitis B Viral Infection (HBV) remains one of the leading cause of morbidity and mortality globally accounting for 38-53% of chronic liver diseases and about 686,000 deaths annually. The prevalence of HBV is 9-20% in Sub-Saharan Africa, and in Kenya it is 5-30% among the general population and 9.4% among pregnant women. This study was aimed at identifying the prevalence, awareness and risk factors associated with HBV infections among pregnant women attending Antenatal clinic (ANC) at Mbagathi District hospital, Nairobi. METHODS: This was a cross-sectional study involving 287 pregnant women enrolled for three months (September to December 2014) from Nairobi and neighbouring counties. A structured questionnaire that captured social, demographic and explanatory variables was administered to the study participants. Blood samples were also drawn from the participants and tested for HBV using Enzyme-Linked Immunosorbent Assay (ELISA) system. RESULTS: The study established that the prevalence of HBV infections among pregnant women attending antenatal clinic at Mbagathi District Hospital was 3.8% with highest infection rate among the 20-24 years age group. Seventy six (60.8 %) of the participants reported sexual encounters in less than a month before the interview of which 5 (7.6%) reported encounters involving other partners apart from their spouses. HBV awareness among the study participants was 12.2%. Before the interview, those with at least tertiary education (Mean =1.33, SD = 1.131), were more informed about HBV infection as compared to those with primary and secondary education (Mean = 0.63, SD = 0.722; (Mean =0.31, SD= 0.664). In regards to assessment of the risk factors; type of family (χ² =19.753 df2 p<0.01), parity (χ² =7.128 df2 p<0.01), History of abortions (χ²=9.094 df1 p<0.01), early age (11-15 years) at first sexual encounter (χ² =8.185 df1 p<0.01) were significantly associated with HBV positivity. CONCLUSION: The prevalence of HBV infection among pregnant women attending Antenatal clinic (ANC) at Mbagathi District hospital, Nairobi was lower (3.8%) than the prevalence among pregnant women nationally (9.4%). These women also showed a low level of HBV awareness (12.2%.).

Genetic divergence of Chikungunya virus plaque variants from the Comoros Island (2005).

Chikungunya virus (CHIKV) from a human sample collected during the 2005 Chikungunya outbreak in the Comoros Island, showed distinct and reproducible large (L2) and small (S7) plaques which were characterized in this study. The parent strain and plaque variants were analysed by in vitro growth kinetics in different cell lines and their genetic similarity assessed by whole genome sequencing, comparative sequence alignment and phylogenetic analysis. In vitro growth kinetic assays showed similar growth patterns of both plaque variants in Vero cells but higher viral titres of S7 compared to L2 in C6/36 cells. Amino acids (AA) alignments of the CHIKV plaque variants and S27 African prototype strain, showed 30 AA changes in the non-structural proteins (nsP) and 22 AA changes in the structural proteins. Between L2 and S7, only two AAs differences were observed. A missense substitution (C642Y) of L2 in the nsP2, involving a conservative AA substitution and a nonsense substitution (R524X) of S7 in the nsP3, which has been shown to enhance O'nyong-nyong virus infectivity and dissemination in Anopheles mosquitoes. The phenotypic difference observed in plaque size could be attributed to one of these AA substitutions. Phylogenetic analysis showed that the parent strain and its variants clustered closely together with each other and with Indian Ocean CHIKV strains indicating circulation of isolates with close evolutionary relatedness in the same outbreak. These observations pave way for important functional studies to understand the significance of the identified genetic changes in virulence and viral transmission in mosquito and mammalian hosts.

Development and Evaluation of an in-House IgM-Capture ELISA for the Detection of Chikungunya and Its Application to a Dengue Outbreak Situation in Kenya in 2013.

Chikungunya (CHIK) is a mosquito-borne viral disease. In the 2004 CHIK outbreak in Kenya, diagnosis was delayed because of the lack of accurate diagnostics. Therefore, this study aimed to develop and evaluate an in-house IgM-capture enzyme linked immunosorbent assay (ELISA) (in-house ELISA) for the detection of chikungunya virus (CHIKV) infections. Anti-CHIKV antibodies were raised in rabbits, purified and conjugated to horseradish peroxidase. These anti-CHIKV antibodies and cell-culture derived antigen were used to develop the ELISA. To validate the in-house ELISA, 148 patient sera from the 2005 Comoros CHIK outbreak were tested with centers for disease control and prevention (CDC) IgM-capture ELISA (CDC ELISA) and focus reduction neutralization test (FRNT) as reference assays. The in-house ELISA had a sensitivity of 97.6% and specificity of 81.3% compared to the CDC ELISA and a sensitivity of 91.1% and specificity of 96.7% compared to FRNT. Furthermore, 254 clinically suspected dengue patient samples from Eastern Kenya, collected in 2013, were tested for CHIKV IgM using the in-house ELISA. Out of the 254 samples, 26 (10.2%) were IgM positive, and of these 26 samples, 17 were further analyzed by FRNT and 14 (82.4%) were positive. The in-house ELISA was able to diagnose CHIKV infection among suspected dengue cases in the 2013 outbreak.

Serological surveillance development for tropical infectious diseases using simultaneous microsphere-based multiplex assays and finite mixture models.

BACKGROUND: A strategy to combat infectious diseases, including neglected tropical diseases (NTDs), will depend on the development of reliable epidemiological surveillance methods. To establish a simple and practical seroprevalence detection system, we developed a microsphere-based multiplex immunoassay system and evaluated utility using samples obtained in Kenya. METHODS: We developed a microsphere-based immuno-assay system to simultaneously measure the individual levels of plasma antibody (IgG) against 8 antigens derived from 6 pathogens: Entamoeba histolytica (C-IgL), Leishmania donovani (KRP42), Toxoplasma gondii (SAG1), Wuchereria bancrofti (SXP1), HIV (gag, gp120 and gp41), and Vibrio cholerae (cholera toxin). The assay system was validated using appropriate control samples. The assay system was applied for 3411 blood samples collected from the general population randomly selected from two health and demographic surveillance system (HDSS) cohorts in the coastal and western regions of Kenya. The immunoassay values distribution for each antigen was mathematically defined by a finite mixture model, and cut-off values were optimized. FINDINGS: Sensitivities and specificities for each antigen ranged between 71 and 100%. Seroprevalences for each pathogen from the Kwale and Mbita HDSS sites (respectively) were as follows: HIV, 3.0% and 20.1%; L. donovani, 12.6% and 17.3%; E. histolytica, 12.8% and 16.6%; and T. gondii, 30.9% and 28.2%. Seroprevalences of W. bancrofti and V. cholerae showed relatively high figures, especially among children. The results might be affected by immunological cross reactions between W. bancrofti-SXP1 and other parasitic infections; and cholera toxin and the enterotoxigenic E. coli (ETEC), respectively. INTERPRETATION: A microsphere-based multi-serological assay system can provide an opportunity to comprehensively grasp epidemiological features for NTDs. By adding pathogens and antigens of interest, optimized made-to-order high-quality programs can be established to utilize limited resources to effectively control NTDs in Africa.

Hepatitis B virus subgenotype A1, occurrence of subgenotype D4, and S gene mutations among voluntary blood donors in Kenya.

Kenya is one of the high endemic zones for hepatitis B virus (HBV) infection. The consensuses on prevalence of the HBV genotypes and the existence of their variants have not been fully established in Kenya. Hence, there is a need to further monitor the diversity of HBV. This study aimed to extend the current molecular and epidemiological information about the geographical distribution of HBV genotypes and subgenotypes, as well as to describe the hepatitis B surface antigen (HBsAg) variants circulating in different Regional Blood Transfusion Centres of Kenya. A total of 32 HBsAg positive blood units from five different blood transfusion centers in Kenya were used in the study. The HBV DNA preS/S-gene was amplified and sequenced. Alignments of S gene were applied using reference sequence from GeneBank. Phylogenetic analysis was performed using the MEGAv4.0 software with the neighbor-joining and maximum composite likelihood methods. Twenty-one plasma samples (65.6%) were DNA positive and were successfully sequenced. Eighteen out of the twenty-one isolates (85.7%) belonged to subgenotype A1 Afro-Asian: six were from Nairobi, four from Kisumu, two from Embu, and three each from Eldoret and Mombasa. The other three strains (14.3%, 3/21) belonged to subgenotype D4 from Mombasa. The HBsAg mutations were detected in nine isolates (42.9%, 9/21). The HBV/A1 and HBV/D4 are dominant among blood donors in Kenya. This demonstrates that continuous monitoring of the HBV diversity would help reveal circulating genotypes and subgenotypes as well as mutants of clinical significance in Kenya.

Genotyping and molecular characterization of hepatitis B virus in liver disease patients in Kenya.

Hepatitis B virus (HBV) genotypes are important in both the clinical manifestation of disease and treatment response. Although Kenya belongs to the African Region (AFR-E) characterized by high mortality and hyperendemicity of HBV, there is a paucity of HBV genotyping data. The aim of this study was to molecularly characterize the basic core promoter/precore (BCP/PC) and complete surface (S) regions of HBV isolated from 61 HBsAg-positive liver disease patients attending Kenyatta National Hospital in Nairobi. HBsAg, HBeAg and viral loads were determined. HBV DNA was amplified and sequenced from 58/61 patients. In addition to the complete genome of two isolates, the BCP/PC and the complete S regions of 43 and 38 isolates, respectively were sequenced. Following phylogenetic analysis of the S region, 38 isolates clustered with subgenotype A1, whereas two isolates clustered with genotype D, one with subgenotype D1 and another as an outlier of the clade containing subgenotype D6 and the D/E recombinant. When the complete genome of the latter isolate was sequenced it clustered with D6. The majority of isolates belonged to serological subtype adw2 and only four to ayw2. Three distinct groups of subgenotype A1, distinguished by different amino acid motifs, circulate in Kenya: two in the African cluster and a monophyletic clade in the "Asian" cluster. HBeAg-negativity was a result of G1896A in genotype D isolates, whereas in subgenotype A1, the HBeAg-negativity was a result of mutations in the Kozak region (1809-1812) or precore start codon (1814-1816). Mutations at positions 1762 and 1764 occurred more frequently in HCC patients (p<0.05). In conclusion, subgenotypes A1, D1 and D6 circulate in liver disease patients in Kenya, with A1 predominating.