Development of an automated amplicon-based next-generation sequencing pipeline for rapid detection of bacteria and fungi directly from clinical specimens.

The timely identification of microbial pathogens is essential to guide targeted antimicrobial therapy and ultimately, successful treatment of an infection. However, the yield of standard microbiology testing (SMT) is directly related to the duration of antecedent antimicrobial therapy as SMT culture methods are dependent on the recovery of viable organisms, the fastidious nature of certain pathogens, and other pre-analytic factors. In the last decade, metagenomic next-generation sequencing (mNGS) has been successfully utilized as a diagnostic tool for various applications within the clinical laboratory. However, mNGS is resource, time, and labor-intensive-requiring extensive laborious preliminary benchwork, followed by complex bioinformatic analysis. We aimed to address these shortcomings by developing a largely Automated targeted Metagenomic next-generation sequencing (tmNGS) PipeLine for rapId inFectIous disEase Diagnosis (AMPLIFIED) to detect bacteria and fungi directly from clinical specimens. Therefore, AMPLIFIED may serve as an adjunctive approach to complement SMT. This tmNGS pipeline requires less than 1 hour of hands-on time before sequencing and less than 2 hours of total processing time, including bioinformatic analysis. We performed tmNGS on 50 clinical specimens with concomitant cultures to assess feasibility and performance in the hospital laboratory. Of the 50 specimens, 34 (68%) were from true clinical infections. Specimens from cases of true infection were more often tmNGS positive compared to those from the non-infected group (82.4% vs 43.8%, respectively, P = 0.0087). Overall, the clinical sensitivity of AMPLIFIED was 54.6% with 85.7% specificity, equating to 70.6% and 75% negative and positive predictive values, respectively. AMPLIFIED represents a rapid supplementary approach to SMT; the typical time from specimen receipt to identification of potential pathogens by AMPLIFIED is roughly 24 hours which is markedly faster than the days, weeks, and months required to recover bacterial, fungal, and mycobacterial pathogens by culture, respectively. IMPORTANCE: To our knowledge, this represents the first application of an automated sequencing and bioinformatics pipeline in an exclusively pediatric population. Next-generation sequencing is time-consuming, labor-intensive, and requires experienced personnel; perhaps contributing to hesitancy among clinical laboratories to adopt such a test. Here, we report a strong case for use by removing these barriers through near-total automation of our sequencing pipeline.

FPGA Implementation of the Chirp-Scaling Algorithm for Real-Time Synthetic Aperture Radar Imaging.

Synthetic aperture radar (SAR), which can generate images of regions or objects, is an important research area of radar. The chirp scaling algorithm (CSA) is a representative SAR imaging algorithm. The CSA has a simple structure comprising phase compensation and fast Fourier transform (FFT) operations by replacing interpolation for range cell migration correction (RCMC) with phase compensation. However, real-time processing still requires many computations and a long execution time. Therefore, it is necessary to develop a hardware accelerator to improve the speed of algorithm processing. In addition, the demand for a small SAR system that can be mounted on a small aircraft or drone and that satisfies the constraints of area and power consumption is increasing. In this study, we proposed a CSA-based SAR processor that supports FFT and phase compensation operations and presents field-programmable gate array (FPGA)-based implementation results. We also proposed a modified CSA flow that simplifies the traditional CSA flow by changing the order in which the transpose operation occurs. Therefore, the proposed CSA-based SAR processor was designed to be suitable for modified CSA flow. We designed the multiplier for FFT to be shared for phase compensation, thereby achieving area efficiency and simplifying the data flow. The proposed CSA-based SAR processor was implemented on a Xilinx UltraScale+ MPSoC FPGA device and designed using Verilog-HDL. After comparing the execution times of the proposed SAR processor and the ARM cortex-A53 microprocessor, we observed a 136.2-fold increase in speed for the 4096 × 4096-pixel image.

Accuracy Validation of the Elecsys HBsAg II Quant Assay and Its Utility in Resolving Equivocal Qualitative HBsAg Results.

Background and Objectives: There are reports of false qualitative HBsAg results, because of various causes, such as samples with low HBsAg concentrations that may produce false positives. The main aims of this study were to validate the analytical accuracy and to assess the utility of the Elecsys assay compared to that of the qualitative HbsAg assay as a screening test in resolving equivocal qualitative HbsAg results. Materials and Methods: The limit of blank (LoB), the limit of detection (LoD), the limit of quantification (LoQ), and linearity were estimated to validate the analytical accuracy of the Elecsys HBsAg II Quant assay. A total of 449 serum samples showing initial equivocal results (1-50 index) were evaluated by Elecsys HBsAg II Quant and ADVIA Centaur HBsAg II assays. Results: The LoQ of the assay was determined to be 0.050 IU/mL, as provided by the manufacturer. The Kappa agreement between the two assays was almost perfect, at 0.9669, despite seven discordant results. With a specificity of 100% at new cut-off index value ≥5.42, about 78 samples (17%, 78/449) with index value ≥5.42 were interpreted as positives without further duplicate tests, however the remaining 371 samples with index value <5.42 need to be confirmed with additional HBV marker assays. Conclusions: We confirm that the Elecsys HBsAg II Quant assay is accurate and sensitive for HBV infection and recommend it as an alternative confirmatory HBsAg assay for resolving equivocal qualitative HBsAg results.

Pyogenic spondylitis caused by Klebsiella pneumoniae: should the possibility of hypervirulent Klebsiella pneumoniae be considered?

BACKGROUND: Klebsiella pneumoniae is rare but the second most common causative agent among gram-negative bacteria that cause pyogenic spondylitis. However, there are no available studies on the serotype, virulence factors, and clinical characteristics associated with K. pneumoniae-caused pyogenic spondylitis. Accordingly, we investigated the clinical characteristics of pyogenic spondylitis, K1 and K2 serotypes, and virulence factors of K. pneumoniae. METHODS: We reviewed the microbiological reports of specimens collected between January 2014 and December 2019 as well as the medical records of patients with pyogenic spondylitis caused by K. pneumoniae. We also evaluated K1 and K2 serotypes and the virulent genes rmpA, iutA, mrkD, ybtS, entB, and kfu. Strains that possessed rmpA and iutA were defined as hypervirulent K. pneumoniae. RESULTS: Six patients with pyogenic spondylitis caused by K. pneumoniae were enrolled in the study. The capsular serotypes K1 and K2 were present in 66.7% (4/6) of cases, and the hypervirulent strains were present in 88.3% (5/6) of cases. All patients had community-acquired infections, and all strains isolated were susceptible to antimicrobial agents. Intravenous antibiotic treatment continued for 2-7 weeks, and no patient underwent decompressive operation or surgical debridement. There was no recurrence. One patient died from pneumonia with a septic lung. CONCLUSION: Hypervirulent K. pneumoniae is a rare but possible causative agent associated with pyogenic spondylitis.

Point-of-care COVID-19 testing: colorimetric diagnosis using rapid and ultra-sensitive ramified rolling circle amplification.

In this paper, we report a molecular diagnostic system-combining a colorimetric probe (RHthio-CuSO4) for pyrophosphate sensing and isothermal gene amplification (ramified rolling circle amplification)-that operates with high selectivity and sensitivity for clinical point-of-care diagnosis of SARS-CoV-2. During the polymerase phase of the DNA amplification process, pyrophosphate was released from the nucleotide triphosphate as a side product, which was then sensed by our RHthio-CuSO4 probe with a visible color change. This simple colorimetric diagnostic system allowed highly sensitive (1.13 copies/reaction) detection of clinical SARS-CoV-2 within 1 h, while also displaying high selectivity, as evidenced by its discrimination of two respiratory viral genomes (human rhino virus and respiratory syncytial virus) from that of SARS-CoV-2. All of the reactions in this system were performed at a single temperature, with positive identification being made by the naked eye, without requiring any instrumentation. The high sensitivity and selectivity, short detection time (1 h), simple treatment (one-pot reaction), isothermal amplification, and colorimetric detection together satisfy the requirements for clinical point-of-care detection of SARS-CoV-2. Therefore, we believe that this combination of a colorimetric probe and isothermal amplification will be useful for point-of-care testing to prevent the propagation of COVID-19.

Phthalate plasticizer decreases the prion-like protein doppel essential for structural integrity and function of spermatozoa.

Di-n-butyl phthalate (DBP), a well-known endocrine disruptor, causes male reproductive dysfunction. To understand the underlying mechanisms, we performed histological, endocrinological, and biochemical analyses and assessed the expression of genes involved in spermatogenesis and sperm function according to OECD test guideline 407. Following 28 days of administration of the lowest observed adverse effect level dose of DBP to mice, no significant changes in body weight, testis and epididymis weights and histology, serum testosterone level, or testicular daily sperm production were found. Nonetheless, the motility of the epididymal sperm of the DBP group was significantly decreased together with an increase in the incidence of bent tails and abnormal heads. In the testes of the DBP group, lipid peroxidation (LPO) level was significantly increased and testicular Bcl-2 mRNA level was significantly decreased together with an increase in the Bax/Bcl-2 mRNA ratio. In the testes of the DBP group, levels of Prnd mRNA and protein and Pou4f1 mRNA, an activator of the Prnd promotor, were significantly decreased. Of note, prion-like protein doppel (PRND) was significantly decreased together with decreased PRND immunoreactivity in the head, midpiece, and tail of sperm. In the testes of the DBP group, levels of Sox9, Sgp1, and Sgp2 mRNA, which are functional Sertoli cell markers, were significantly decreased. Level of Amh mRNA, a Sertoli cell immaturity marker, was significantly increased together with that of Inha mRNA, suggesting deregulation of the brain-gonadal axis. Together, our findings suggest that DBP at present dosage may potentiate LPO generation and Sertoli cell immaturity via downregulation of Sox9 and disruption of the Pou4f1-Prnd gene network in post-meiotic germ cells without visible changes in spermatogenesis or testosterone level. This may result in structural and functional abnormalities in spermatozoa. Additionally, our findings suggest that assessment of the male reproductive toxicity of phthalate ester plasticizers based on conventional OECD test guidelines should be reconsidered.

Two Case Reports of Life-Threatening Croup Caused by the SARS-CoV-2 Omicron BA.2 Variant in Pediatric Patients.

Croup is a common upper airway infection characterized by a barking cough, stridor, and hoarseness. It is usually caused by viral infection. A small number of croup caused by coronavirus disease 2019 (COVID-19) has been reported in children before the omicron variant surge. Previously reported cases indicated that croup caused by COVID-19 can be treated in the same manner as those with other viral causes. We describe two cases (9-month-old girl and 11-month-old boy) of previously healthy infants who presented with a barking cough and chest retraction and required endotracheal intubation and cardiopulmonary resuscitation. Despite receiving dexamethasone and nebulized racemic epinephrine (NRE) treatment for croup in the emergency department, these patients still developed acute respiratory failure. Reverse transcription polymerase chain reaction (RT-PCR) of nasopharyngeal samples revealed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) omicron BA.2 variant (Stealth omicron) and no other common respiratory viral pathogens. Both patients were treated with mechanical ventilation, dexamethasone, and NRE in the pediatric intensive care unit. The duration of intubation was 112 hours and 80 hours, respectively. Both patients were discharged without complications. To the best of our knowledge, this is the first report of life-threatening croup produced by the omicron BA.2 variant and confirmed by RT-PCR. We suggest that this SARS-CoV-2 variant may cause severe croup that may not improve with conventional treatment, even in children without underlying diseases.

Dual-site ligation-assisted loop-mediated isothermal amplification (dLig-LAMP) for colorimetric and point-of-care determination of real SARS-CoV-2.

A probing system has been developed based on dual-site ligation-assisted loop-mediated isothermal amplification (dLig-LAMP) for the selective colorimetric detection of SARS-CoV-2. This approach can induce false-positive and -negative detection in real clinical samples; dLig-LAMP operates with improved selectivity. Unlike RT-LAMP, the selectivity of dLig-LAMP is determined in both the ligation and primer binding steps, not in the reverse transcription step. With this selective system in hand, we developed a colorimetric signaling system for point-of-care detection. We also developed a colorimetric probe for sensing pyrophosphate, which arises as a side product during the LAMP DNA amplification. Thus, dLig-LAMP appears to be an alternative method for improving the selectivity problems associated with reverse transcription. In addition, combining dLig-LAMP with colorimetric pyrophosphate probing allows point-of-care detection of SARS-CoV-2 within 1 h with high selectivity.

Invasive Hypervirulent Klebsiella pneumoniae Syndrome Originating from an Anorectal Abscess as Opposed to a Pyogenic Liver Abscess.

An immunocompetent 49-year-old man presented with swelling and pain in the lower region of his left leg that had lasted for 4 weeks. The diagnosis was severe pyomyositis and osteomyelitis in the lower left leg caused by hypervirulent Klebsiella pneumoniae (hvKP) along with multiple metastatic infections in the kidneys, lungs, and brain originating from an anorectal abscess. A virulence-gene analysis revealed that the isolated K. pneumoniae harbored rmpA, entB, ybtS, kfu, iutA, mrkD, and allS-virulence genes and belonged to the K1 capsular serotype. After repeated abscess drainage procedures, intravenous ceftriaxone was administered for more than 10 weeks, and the patient's infection was controlled. We focused on the clinical features of hvKP originating from an anorectal abscess without a pyogenic liver abscess. We suggest that hvKP be considered a causative pathogen of pyomyositis and osteomyelitis resulting in multiple metastatic infections in an immunocompetent patient, and more information on the unexpected multiple metastatic infections should be obtained from a virulence analysis of K. pneumoniae.

Evidence of Severe Acute Respiratory Syndrome Coronavirus 2 Reinfection After Recovery from Mild Coronavirus Disease 2019.

BACKGROUND: Positive results from real-time reverse-transcription polymerase chain reaction (rRT-PCR) in recovered patients raise concern that patients who recover from coronavirus disease 2019 (COVID-19) may be at risk of reinfection. Currently, however, evidence that supports reinfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has not been reported. METHODS: We conducted whole-genome sequencing of the viral RNA from clinical specimens at the initial infection and at the positive retest from 6 patients who recovered from COVID-19 and retested positive for SARS-CoV-2 via rRT-PCR after recovery. A total of 13 viral RNAs from the patients' respiratory specimens were consecutively obtained, which enabled us to characterize the difference in viral genomes between initial infection and positive retest. RESULTS: At the time of the positive retest, we were able to acquire a complete genome sequence from patient 1, a 21-year-old previously healthy woman. In this patient, through the phylogenetic analysis, we confirmed that the viral RNA of positive retest was clustered into a subgroup distinct from that of the initial infection, suggesting that there was a reinfection of SARS-CoV-2 with a subtype that was different from that of the primary strain. The spike protein D614G substitution that defines the clade "G" emerged in reinfection, while mutations that characterize the clade "V" (ie, nsp6 L37F and ORF3a G251V) were present at initial infection. CONCLUSIONS: Reinfection with a genetically distinct SARS-CoV-2 strain may occur in an immunocompetent patient shortly after recovery from mild COVID-19. SARS-CoV-2 infection may not confer immunity against a different SARS-CoV-2 strain.

N-acetylcysteine reduce the stress induced by cold storage of platelets: A potential way to extend shelf life of platelets.

The room temperature storage used for platelets worldwide leads to platelet storage lesion (PSL) and risk of bacterial growth, limiting platelet shelf life and safety in transfusion. Thus, there is a need for an alternative storage method that can serve as effective temperature storage for platelet concentrates (PCs). In the previous investigation, we have shown that N-acetylcysteine (NAC) is a potential candidate for an additive solution to retain platelet characteristics during cold storage for up to 5 days. However, the study partially describes the efficacy and has drawbacks to address. Here, we used the apheresis platelet product with 50 mM NAC and stored up to 10 days under refrigerated condition (4 ± 1 °C). Stored platelet concentrates were analyzed for critical parameters such as platelet activation, annexin V binding, sialic acid, reactive oxygen species (ROS), neuraminidase activity, and in vivo efficacy using Prkdcscid mice. Investigation observations revealed that PCs with NAC showed reduced platelet activation, annexin V binding, ROS production, and sialic acid levels. in vivo recovery of PCs showed similar recovery rates stored PCs irrespective of treatment or storage condition. However, on the tenth day after 24 h, recovery in room temperature stored concentrates was about 32 %, whereas in NAC treated refrigerated concentrates, it stands at 47 %. These observations indicate that NAC addition protects refrigerated concentrates during long-term storage retaining the platelet integrity. The study also suggests that extending PC storage beyond 10 days is practically accomplishable with efficacy similar to room temperature (RT) stored PCs.

Analytical Performance of the Sysmex HISCL HBsAg Assay and Comparison with the Roche Elecsys HBsAg II Quant Assay in the Quantification of Hepatitis B Surface Antigen.

Background and Objectives: This study aims to estimate the analytical performance of the Sysmex HISCL HBsAg assay and to assess the analytical correlation with the Roche Elecsys HBsAg II quant assay with clinical samples and the WHO International Standard (IS). Materials and Methods: The intra-assay precision, linearity, assay limitation, accuracy, and comparative evaluation of the HISCL HBsAg assay were estimated. Results: Extrapolating from the plot of the average total allowable error versus the reference value, an accuracy goal of 20% would be achieved around a limit of quantification (LoQ) of 0.014867 IU/mL. The percentage of biases for each level of the WHO IS measured by the two assays were less than 15%, except for the WHO 3rd IS, for which the HISCL HBsAg assay achieved a percentage of bias of 33%. In the comparative evaluation, Passing-Bablok regression analysis did not reveal any significant deviation from linearity between the two assays (y = -48.6998 + 1.9206x; p = 0.79 by the CUSUM test for linearity). The mean difference of the quantitative HBsAg level between the two assays was 1762.5 IU/mL in the Bland-Altman plot. Conclusions: The HISCL HBsAg assay, with a highly sensitive LoQ of 0.03 IU/mL, showed similar analytical performance in HBsAg quantification to the Elecsys HBsAg II quant assay and may be helpful in obtaining better diagnoses and therapeutic strategies for treating HBV infections.

Pooling Upper Respiratory Specimens for Rapid Mass Screening of COVID-19 by Real-Time RT-PCR.

To validate the specimen-pooling strategy for real-time reverse transcription PCR detection of severe acute respiratory syndrome coronavirus 2, we generated different pools including positive specimens, reflecting the distribution of cycle threshold values at initial diagnosis. Cumulative sensitivities of tested pool sizes suggest pooling of <6 specimens for surveillance by this method.

Pressure-driven spontaneous ion concentration polarization using an ion-selective membrane.

In this study, the spontaneous ion concentration polarization phenomenon induced by pressure via a cation-selective membrane was theoretically and experimentally investigated. Unlike conventional electrokinetic ion concentration polarization, which uses electric current as a driving flux of cations through the membrane, advection caused by pressure is used as a transmembrane driving flux of cations to spontaneously and stably form an ion depletion zone in the present ion concentration polarization technique. The ion depletion zone produced in a simple experimental setup was used to filter electrolyte and preconcentrate ions and microparticles. Different from the general assumption of the negligible thickness of the electric double layer in microchannels, the low concentration in the ion depletion zone considerably increased the length of the electric double layer. This enhanced the formation of the ion depletion zone. The present results can improve the understanding on ion transport in the ion concentration polarization system and can be utilized to develop a portable water desalination device for rural/remote areas and for preconcentrating biomolecules.

Comparative study of idursulfase beta and idursulfase in vitro and in vivo.

Hunter syndrome is an X-linked lysosomal storage disease caused by a deficiency in the enzyme iduronate-2-sulfatase (IDS), leading to the accumulation of glycosaminoglycans (GAGs). Two recombinant enzymes, idursulfase and idursulfase beta are currently available for enzyme replacement therapy for Hunter syndrome. These two enzymes exhibited some differences in various clinical parameters in a recent clinical trial. Regarding the similarities and differences of these enzymes, previous research has characterized their biochemical and physicochemical properties. We compared the in vitro and in vivo efficacy of the two enzymes on patient fibroblasts and mouse model. Two enzymes were taken up into the cell and degraded GAGs accumulated in fibroblasts. In vivo studies of two enzymes revealed similar organ distribution and decreased urinary GAGs excretion. Especially, idursulfase beta exhibited enhanced in vitro efficacy for the lower concentration of treatment, in vivo efficacy in the degradation of tissue GAGs and improvement of bones, and revealed lower anti-drug antibody formation. A biochemical analysis showed that both enzymes show largely a similar glycosylation pattern, but the several peaks were different and quantity of aggregates of idursulfase beta was lower.

Identification and Validation of SAA4 as a Rheumatoid Arthritis Prescreening Marker by Liquid Chromatography Tandem-mass Spectrometry.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that progresses into systemic inflammation and joint deformity. RA diagnosis is a complicated procedure, and early diagnostic methods are insufficient. Therefore, in this study, we attempted to identify new markers to improve the accuracy of RA prescreening. e identified differentially expressed proteins (DEPs) by using liquid chromatography tandem-mass spectrometry in health-prescreening sera with high rheumatoid factor (RF) values, and compared the findings with those from sera with normal RF values. We identified 93 DEPs; of these, 36 were upregulated, and 57 were downregulated in high-RF sera. Pathway analysis revealed that these DEPs were related to immune responses. Additionally, four DEPs were statistically analyzed by proteomic analysis; of these, SAA4 was significantly validated in individual enzyme-linked immunosorbent assays. Moreover, SAA4 was significantly upregulated in RA patients (n = 40, 66.43 ± 12.97 ng/mL) compared with normal controls (n = 40, 4.79 ± 0.95 ng/mL) and had a higher area under the curve than C-reactive protein. Thus, we identified SAA4 as a protein that was positively correlated with RF and RA. SAA4 may represent a novel prescreening marker for the diagnosis of RA.

Evaluation of in vitro storage characteristics of cold stored platelet concentrates with N acetylcysteine (NAC).

Platelets play a vital role in hemostasis and thrombosis, and their demand and usage has multiplied many folds over the years. However, due to the short life span and storage constraints on platelets, it is allowed to store them for up to 7 days at room temperature (RT); thus, there is a need for an alternative storage strategy for extension of shelf life. Current investigation involves the addition of 50 mM N acetylcysteine (NAC) in refrigerated concentrates. Investigation results revealed that addition of NAC to refrigerated concentrates prevented platelet activation and reduced the sialidase activity upon rewarming as well as on prolonged storage. Refrigerated concentrates with 50 mM NAC expressed a 23.91 ± 6.23% of CD62P (P-Selectin) and 22.33 ± 3.42% of phosphotidylserine (PS), whereas RT-stored platelets showed a 46.87 ± 5.23% of CD62P and 25.9 ± 6.48% of phosphotidylserine (PS) after 5 days of storage. Further, key metabolic parameters such as glucose and lactate accumulation indicated reduced metabolic activity. Taken together, investigation and observations indicate that addition of NAC potentially protects refrigerated concentrates by preventing platelet activation, stabilizing sialidase activity, and further reducing the metabolic activity. Hence, we believe that NAC can be a good candidate for an additive solution to retain platelet characteristics during cold storage and may pave the way for extension of storage shelf life.

A taxonomic study of four rare pteromalid genera: Amblyharma Huang & Tong, Fusta Xiao & Ye, Nazgulia Hedqvist and Platecrizotes Ferrière from the Eastern Palaearctic (Chalcidoidea, Pteromalidae, Pachyneurinae).

The four morphologically similar genera Amblyharma Huang & Tong, 1993, Fusta Xiao & Ye, 2015, Nazgulia Hedqvist, 1973 and Platecrizotes Ferrière, 1934 from the Eastern Palaearctic are reviewed. Redescriptions of genera and all available types of Eastern Palaearctic species are provided. An identification key to genera is given. A new species from South Korea, Platecrizotesjediisp. nov. is described and illustrated.

Smallholders' vulnerability in the maize market: An analysis of marketing channels to improve the role of cooperatives in Benin.

Smallholder households in developing countries often face challenges related to market access. Despite being proposed as a potential solution, cooperatives in the north of Benin have reportedly failed to effectively address this issue. The alternatives channels used by smallholder famers and the factors that influence their choices remain unclear. This paper adopts a mixed approach, combining qualitative analysis with quantitative methods, particularly the multivariate probit model, to investigate the distribution channels of maize used by farmers in the Kandi district of Benin. The study aims to identify the current distribution channels of maize in Kandi and analyze factors that affect, smallholder producers choice of marketing channel. Initially, the study identified four primary channels for marketing maize-namely, collectors, wholesalers, brokers, and cooperatives-and producers typically commonly engage in multiple channels simultaneously. Subsequently, It becomes evident that factors such as age, cooperative membership, distance to market, reliance on informal credit, average maize bag prices, and the timing of sales significantly influence producers' selection of marketing channels. Interestingly, despite cooperatives offering comparatively higher prices, the majority of farmers opt for collectors due to the informal credit they provide and their practice of purchasing large volumes of maize bags at pre-agreed prices. In conclusion, enhancing joint-selling capabilities of cooperatives and establishing a credit provision business tailored to maize producers are identified as crucial steps. These measures aim to alleviate producers' financial strain, diminish their dependence on collectors for credit, and bolster their bargaining power in the market.