Predictors of lung injury during durvalumab maintenance therapy following concurrent chemoradiotherapy in unresectable locally advanced non-small cell lung carcinoma.

BACKGROUND: Based on the results of the PACIFIC trial, maintenance with durvalumab has emerged as the standard treatment following concurrent chemoradiotherapy in patients with unresectable locally advanced non-small cell lung carcinoma (NSCLC). However, adverse events attributed to durvalumab, especially lung injuries, including immune-related adverse events, and radiation pneumonitis, are concerning. This study retrospectively investigated the factors related to lung injury in patients receiving the PACIFIC regimen. METHODS: Patients with unresectable locally advanced NSCLC who received durvalumab maintenance therapy following concurrent chemoradiotherapy at Yokohama City University Medical Centre between July 2018 and March 2022 were included. Clinical data, volume of normal lung receiving 20 or 5 Gy or more (V20 or V5), planning target volume (PTV), and relative lung parenchyma volume in emphysematous lung receiving 20 or 5 Gy or more (RLPV20 or 5; V20 or V5/100-percentage of low-attenuation volume) were evaluated. RESULTS: Performance status (PS), V20, V5, PTV, RLPV20, and RLPV5 were significantly higher in the lung injury group in the univariate analysis. Furthermore, RLPV20 was the most significant factor in the lung injury group in the multivariate analysis comprising PS, PTV, V20, and RLPV20. CONCLUSION: RLPV20 and RLPV5 are useful in estimating lung inflammation. RLPV20 could be considered the most reliable risk factor for maintenance therapy with durvalumab following concurrent chemoradiotherapy in patients with unresectable locally advanced NSCLC.

Thymic atypical carcinoid tumors with elevated mitotic counts in a patient with multiple endocrine neoplasia: A case report.

Thymic neuroendocrine tumors associated with multiple endocrine neoplasia are only defined as carcinoid and are not associated with large-cell neuroendocrine carcinoma (LCNEC). We report the case of a multiple endocrine neoplasia type 1 patient with atypical carcinoid tumors with elevated mitotic counts (AC-h), an intermediate condition between carcinoid and LCNEC. A 27-year-old man underwent surgery for an anterior mediastinal mass and was diagnosed with thymic LCNEC. Fifteen years later, a mass appeared at the same site, which was determined to be a postoperative recurrence based on the pathological results of a needle biopsy and the clinical course. The patient's disease remained stable for 10 months on anti-programmed death-ligand 1 antibody and platinum-containing chemotherapy. The needle biopsy specimen was submitted for next-generation sequencing, which revealed a MEN1 gene mutation, and after further examination, a diagnosis of multiple endocrine neoplasia type 1 was made. A re-examination of the surgical specimen from 15 years prior showed that it corresponded to AC-h. Although thymic AC-h is classified as thymic LCNEC according to the current definition, our data suggests that a search for multiple endocrine neoplasia is warranted in such patients.

Primary tracheal small-cell carcinoma detected 11 months after surgery for pulmonary large-cell neuroendocrine carcinoma: A case report.

Primary tracheal small-cell carcinoma is rare, and is often treated using small-cell lung cancer guidelines given that no standard treatment has been established for it. We report a patient in whom nodules appeared in the trachea and left main bronchus 11 months after surgery for pulmonary large-cell neuroendocrine carcinoma; a biopsy revealed small-cell carcinoma. Given the absence of malignant lesions elsewhere in the body, the lesions were diagnosed as primary tracheal small-cell carcinoma. Respiratory failure progressed rapidly owing to airway stenosis caused by the growing lesion, and the patient required nasal high-flow therapy. However, the lesions shrank a few days after commencing first-line chemotherapy, and his respiratory failure resolved. Accelerated hyperfractionated radiotherapy was administered in conjunction with the third course of chemotherapy, and the patient ultimately achieved a complete response. Although the lesions were initially suspected of being postoperative recurrence of pulmonary large-cell neuroendocrine carcinoma, the fact that the biopsy revealed them to be primary tracheal small-cell carcinoma indicates that intra-airway nodules that appear after lung cancer surgery may possibly be primary tracheal tumors.

Retrospective analysis of independent predictors of progression-free survival in patients with EGFR mutation-positive advanced non-small cell lung cancer receiving first-line osimertinib.

BACKGROUND: Clinically measurable factors affecting the progression-free survival (PFS) of patients receiving osimertinib as first-line therapy for epidermal growth factor receptor (EGFR) mutation-positive advanced non-small cell lung cancer (NSCLC) have not yet been established. METHODS: We retrospectively reviewed the medical records of 61 patients treated with osimertinib as primary therapy for EGFR mutation-positive advanced NSCLC at Yokohama City University Medical Center between August 2018 and March 2022. Our objective was to identify the independent predictors of PFS. RESULTS: The median age of participants was 74 years. Overall, 73.8% had good (0-1) Eastern Cooperative Oncology Group performance status (PS), and 98.4% had histology of adenocarcinoma. The EGFR mutation was exon19 deletion in 52.5% and exon21 L858R in 44.3% of patients. Programmed death-ligand 1 tumor proportion score >50% was observed in 21.3% and liver metastasis in 9.9% of patients. Median PFS was 19.5 months (95% confidence interval [CI]: 10.6-31.6), and overall survival was not reached. The objective response rate was 68.9%, and disease control rate was 93.4%. Multivariate analysis showed that poor PS (2-4) negatively impacted PFS (hazard ratio, 3.79; 95% CI: 1.46-9.87; p = 0.006). Median PFS in the good PS and poor PS groups was 20.4 months (95% CI: 12.4-not evaluable) and 7.2 months (95% CI: 7.2-19.5), respectively. Interstitial lung disease of all grades and grade 3 was observed as an adverse event in 6.6 and 4.9% of patients, respectively. CONCLUSION: Poor PS was associated with poor prognosis in patients with EGFR mutation-positive advanced NSCLC treated with osimertinib as first-line therapy.

Molecular identification and risk factor analysis of the first Lumpy skin disease outbreak in cattle in Mongolia.

Lumpy skin disease (LSD) is a transboundary viral infectious disease in cattle caused by a Capripoxvirus. LSD has been recently introduced in some Asian countries. However, in Mongolia, no report of LSD is publicly available. We clinically examined LSD symptoms in 1,034 cattle from 4 soum (district) in Dornod province in Mongolia. Sixty-one cattle of them were confirmed with symptoms of LSD and then viral P32 gene was detected by a PCR. The overall prevalence of LSD in cattle was 5.9%. Females odds ratios (OR)=2.27 than males, adults (>2.5-years-old, OR=3.68) than young (1-2.5-years-old) and calves (<1-year-old) were at higher risks for LSD cases in Mongolia, while locations near the tube well and pond water are major risk areas for viral transmission due to density of insects often is high. For virus isolation, skin nodule tissue samples of 4 cattle located in four distinct soums were used for viral propagation using the MDBK cell line. Internal terminal repeat region and RPO30 gene of 4 Mongolian isolates were amplified and sequenced. In the phylogenetic trees, Mongolian LSDVs (2021) were clustered together with the Chinese (2020) and Vietnamese isolates (2020). This is the first report alarming the LSD outbreak in Mongolia that was confirmed by our study. The newly isolated viruses would be a useful base for developing diagnostic tools and inactivated vaccine technology. A large-scale study of LSD is next priority for establishing successful control strategy of further disease outbreak.

Combination of Pembrolizumab With Platinum-containing Chemotherapy for Pulmonary Enteric Adenocarcinoma.

Background/Aim: Pulmonary enteric adeno-carcinoma (PEAC) is a rare type of non-small cell lung cancer (NSCLC), for which no established standard treatment exists. Combination therapy with the anti-programmed cell death protein 1 antibody pembrolizumab and platinum-containing chemotherapy is the standard treatment for NSCLC patients, but its effectiveness in PEAC is uncertain. Case Report: We present a 68-year-old man with chemotherapy-naïve advanced PEAC who responded to a combination of pembrolizumab and platinum-containing chemotherapy. Conclusion: The number of PEAC cases is small, and no clinical trials have been conducted to determine an optimal chemotherapy regimen. In this case, we showed that pembrolizumab combined with platinum-containing chemotherapy might effectively treat PEAC.

Genotyping of Theileria parva populations in vaccinated and non-vaccinated cattle in Malawi.

Theileria parva is an apicomplexan protozoan parasite that causes bovine theileriosis (East Coast Fever; ECF) in central, eastern and southern Africa. In Malawi, ECF is endemic in the northern and central regions where it has negatively affected the development of dairy industry. Despite its endemic status the genetic population structure of T. parva in Malawi is currently unknown. To obtain an understanding of T. parva in Malawi, we performed population genetics analysis of T. parva populations in cattle vaccinated with the Muguga cocktail live vaccine and non-vaccinated cattle using mini- and microsatellite markers covering all the four T. parva chromosomes. The T. parva Muguga strain was included in this study as a reference strain. Linkage disequilibrium was observed when all samples were treated as a single population. There was sub-structuring among the samples as shown by the principal coordinate analysis. Majority of the samples clustered with the T. parva Muguga reference strain suggesting that the isolates in Malawi are closely related to the vaccine component, which support the current use of Muguga cocktail vaccine to control ECF. The clustering of samples from non-endemic southern region with those from endemic central region suggests expansion of the distribution of T. parva in Malawi.

High infection rate of tick-borne protozoan and rickettsial pathogens of cattle in Malawi and the development of a multiplex PCR for Babesia and Theileria species identification.

Malawi has an estimated cattle population of 1,884,803 heads, the indigenous Malawi zebu breed accounts for 91.2%, while the exotic and crossbred accounts for the remaining 8.8%. Although ticks and tick-borne diseases are widespread in Malawi, no molecular study has been conducted to investigate the tick-borne Anaplasmataceae and piroplasms infecting cattle. To provide an insight into the current status of tick-borne pathogens (TBPs) of cattle, a molecular survey was conducted in the central and southern regions of Malawi. A total of 191 cattle of which 132 were Malawi zebu, 44 were Holstein Friesian and 15 were Holstein-Friesian/ Malawi zebu crosses were screened for Anaplasmataceae and piroplasms using the heat shock protein groEL gene and 18S rDNA, respectively. A new 18S rDNA multiplex PCR assay was designed for Babesia and Theileria species identification without sequencing. Overall, 92.3% (n = 177) of the examined animals were infected with at least one TBP. Anaplasmataceae-positive rate was 57.6% (n = 110) while for piroplasms it was 80.1% (n = 153). The detected Anaplasmataceae were Anaplasma bovis 2.6% (n = 5), Anaplasma marginale 24.6% (n = 47), Anaplasma platys-like 13.6% (n = 26), uncharacterized Anaplasma sp. 14.1% (n = 27), and uncharacterized Ehrlichia sp. 16.2% (n = 31). The detected piroplasms were Babesia bigemina 2.6% (n = 5), Theileria mutans 73.8% (n = 141), Theileria parva 33.0% (n = 63), Theileria taurotragi 12.6% (n = 24), and Theileria velifera 53.4% (n = 102). Mixed infection rate was found in 79.6% (n = 152) of the samples analyzed. This study has shown a high burden of TBPs among cattle in Malawi which highlights the need to conceive new methods to control ticks and TBPs in order to improve animal health and productivity. The newly developed multiplex PCR assay would be a useful tool especially in resource limited settings where sequencing is not available and when mixed infections are expected.

Immunization Coverage and Antibody Retention against Rabies in Domestic Dogs in Lusaka District, Zambia.

Rabies remains endemic in Zambia. Despite conducting canine vaccinations in Lusaka district, the vaccination coverage and actual seropositivity in the dog population in Lusaka district are rarely evaluated. This study estimated the seropositivity-based immunization coverage in the owned dog population in Lusaka district using the expanded program on immunization cluster survey method. The time-series trend of neutralizing antibodies against rabies in vaccinated dogs was also evaluated. Of 366 dogs in 200 dog-owning households in Lusaka district, blood samples were collected successfully from 251 dogs. In the sampled dogs, 42.2% (106/251) had an antibody titer ≥0.5 IU/mL. When the 115 dogs whose blood was not collected were assumed to be seronegative, the minimum immunization coverage in Lusaka district's owned dog population was estimated at 29.0% (95% confidence interval: 22.4-35.5). It was also found that a single vaccination with certified vaccines is capable of inducing protective levels of antibodies. In contrast, higher antibody titers were observed in multiple-vaccinated dogs than in single-vaccinated dogs, coupled with the observation of a decline in antibody titer over time. These results suggest the importance of continuous booster immunization to maintain herd immunity and provide useful information to plan mass vaccination against rabies in Zambia.

Molecular Detection and Characterization of Rickettsia asembonensis in Human Blood, Zambia.

Rickettsia asembonensis is a flea-related Rickettsia with unknown pathogenicity to humans. We detected R. asembonensis DNA in 2 of 1,153 human blood samples in Zambia. Our findings suggest the possibility of R. asembonensis infection in humans despite its unknown pathogenicity.

Screening of tick-borne pathogens in argasid ticks in Zambia: Expansion of the geographic distribution of Rickettsia lusitaniae and Rickettsia hoogstraalii and detection of putative novel Anaplasma species.

Ticks (Ixodidae and Argasidae) are important arthropod vectors of various pathogens that cause human and animal infectious diseases. Many previously published studies on tick-borne pathogens focused on those transmitted by ixodid ticks. Although there are increasing reports of viral pathogens associated with argasid ticks, information on bacterial pathogens they transmit is scarce. The aim of this molecular study was to detect and characterize Rickettsia and Anaplasmataceae in three different argasid tick species, Ornithodoros faini, Ornithodoros moubata, and Argas walkerae collected in Zambia. Rickettsia hoogstraalii and Rickettsia lusitaniae were detected in 77 % (77/100) of Ar. walkerae and 10 % (5/50) of O. faini, respectively. All O. moubata pool samples (n = 124) were negative for rickettsial infections. Anaplasmataceae were detected in 63 % (63/100) of Ar. walkerae and in 82.2 % (102/124) of O. moubata pools, but not in O. faini. Phylogenetic analysis based on the concatenated sequences of 16S rRNA and groEL genes revealed that Anaplasma spp. detected in the present study were distinct from previously validated Anaplasma species, indicating that the current knowledge on the diversity and vector range of Anaplasma spp. is incomplete. Our findings highlight new geographical records of R. lusitaniae and R. hoogstraalii and confirm that the wide geographic distribution of these species includes the African continent. The data presented here increase our knowledge on argasid tick-borne bacteria and contribute toward understanding their epidemiology.

Domestic dog demographics and estimates of canine vaccination coverage in a rural area of Zambia for the elimination of rabies.

BACKGROUND: An estimated 75% or more of the human rabies cases in Africa occur in rural settings, which underscores the importance of rabies control in these areas. Understanding dog demographics can help design strategies for rabies control and plan and conduct canine mass vaccination campaigns effectively in African countries. METHODOLOGY/PRINCIPAL FINDINGS: A cross-sectional survey was conducted to investigate domestic dog demographics in Kalambabakali, in the rural Mazabuka District of Zambia. The population of ownerless dogs and the total achievable vaccination coverage among the total dog population was estimated using the capture-recapture-based Bayesian model by conducting a canine mass vaccination campaign. This study revealed that 29% of the domestic dog population was under one year old, and 57.7% of those were under three months old and thus were not eligible for the canine rabies vaccination in Zambia. The population growth was estimated at 15% per annum based on the cross-sectional household survey. The population of ownerless dogs was estimated to be small, with an ownerless-to-owned-dog ratio of 0.01-0.06 in the target zones. The achieved overall vaccination coverage from the first mass vaccination was estimated 19.8-51.6%. This low coverage was principally attributed to the owners' lack of information, unavailability, and dog-handling difficulties. The follow-up mass vaccination campaign achieved an overall coverage of 54.8-76.2%. CONCLUSIONS/SIGNIFICANCE: This paper indicates the potential for controlling canine rabies through mass vaccination in rural Zambia. Rabies education and responsible dog ownership are required to achieve high and sustainable vaccination coverage. Our findings also propose including puppies below three months old in the target population for rabies vaccination and emphasize that securing an annual enforcement of canine mass vaccination that reaches 70% coverage in the dog population is necessary to maintain protective herd immunity.

Genetic Diversity of African Trypanosomes in Tsetse Flies and Cattle From the Kafue Ecosystem.

We clarified the genetic diversity of Trypanosoma spp. within the Kafue ecosystem, using PCR targeting the internal transcribed spacer 1 and the cathepsin L-like cysteine protease (CatL) sequences. The overall prevalence of Trypanosoma spp. in cattle and tsetse flies was 12.65 and 26.85%, respectively. Cattle positive for Trypanosoma vivax had a significantly lower packed cell volume, suggesting that T. vivax is the dominant Trypanosoma spp. causing anemia in this area. Among the 12 operational taxonomic units (OTUs) of T. vivax CatL sequences detected, one was from a known T. vivax lineage, two OTUs were from known T. vivax-like lineages, and nine OTUs were considered novel T. vivax-like lineages. These findings support previous reports that indicated the extensive diversity of T. vivax-like lineages. The findings also indicate that combining CatL PCR with next generation sequencing is useful in assessing Trypanosoma spp. diversity, especially for T. vivax and T. vivax-like lineages. In addition, the 5.42% prevalence of Trypanosoma brucei rhodesiense found in cattle raises concern in the community and requires careful monitoring of human African trypanosomiasis.

Molecular identification and genetic characterization of tick-borne pathogens in sheep and goats at two farms in the central and southern regions of Malawi.

Tick-borne diseases (TBDs) caused by pathogens belonging to the genera Anaplasma, Ehrlichia, Babesia and Theileria in small ruminants are widespread in the tropical and sub-tropical countries. The epidemiology of tick-borne pathogens (TBPs) in small ruminants is less understood compared to those infecting cattle in general. This study was carried out to investigate and characterize TBPs in sheep and goats using molecular tools. A total of 107 blood samples from sheep (n = 8) and goats (n = 99) were collected from animals that were apparently healthy from two farms in the central and the southern regions of Malawi. The V4 hypervariable region of the 18S ribosomal RNA gene (rDNA) and the V1 hypervariable region of the 16S rDNA polymerase chain reaction (PCR) assays were used for detection of tick-borne piroplasms and Anaplasmataceae, respectively. Almost the full-length 18S rDNA and the heat shock protein (groEL) gene sequences were used for genetic characterization of the piroplasms and Anaplasmataceae, respectively. The results showed that 76.6 % of the examined animals (n = 107) were positive for at least one TBP. The overall co-infection with at least two TBPs was observed in forty-eight animals (45 %). The detected TBPs were Anaplasma ovis (65 %), Ehrlichia ruminantium (4%), Ehrlichia canis (2%), Babesia strain closely related to Babesia gibsoni (1%), Theileria ovis (52 %), Theileria mutans (3%), Theileria separata (2%), Anaplasma sp. (1%) and Theileria sp. strain MSD-like (17 %). To the authors knowledge this is the first molecular study of TBPs in sheep and goats in Malawi. These results have therefore provided a significant milestone in the knowledge of occurrence of TBPs in sheep and goats in Malawi, which is prerequisite to proper diagnosis and control.

Molecular detection and characterization of tick-borne hemoparasites and Anaplasmataceae in dogs in major cities of Malawi.

Tick-borne pathogens (TBPs) in dogs have attracted much attention over the last decade since some are now known to be zoonotic and pose a threat to both animal and human health sectors. Despite the increase in the number of studies on canine TBPs worldwide, only a few studies have been conducted in resource-limited countries where research priority is given to food animals than companion animals. In the present study, the occurrence of TBPs of the genera Babesia, Hepatozoon, Anaplasma, and Ehrlichia was investigated in 209 owned and stray dogs in three major cities in Malawi through molecular techniques. Among the examined dogs, 93 (44.5%) were infected with at least one TBP. The detection rates were 23.1% for Babesia rossi, 2.9% for B. vogeli, 19.1% for Hepatozoon canis, 2.4% for Anaplasma platys, and 3.8% for Ehrlichia canis. This is the first molecular study that has provided evidence that dogs in Malawi are infected with TBPs. Sensitization is required for veterinary practitioners, dog handlers, and pet owners as the detected pathogens affect the animals' wellbeing. Further studies focusing on rural areas with limited or no access to veterinary care are required to ascertain the extent of the TBP infection in dogs.

Investigation of the piroplasm diversity circulating in wildlife and cattle of the greater Kafue ecosystem, Zambia.

BACKGROUND: Piroplasms are vector-borne intracellular hemoprotozoan parasites that infect wildlife and livestock. Wildlife species are reservoir hosts to a diversity of piroplasms and play an important role in the circulation, maintenance and evolution of these parasites. The potential for likely spillover of both pathogenic and non-pathogenic piroplasm parasites from wildlife to livestock is underlined when a common ecological niche is shared in the presence of a competent vector. METHOD: To investigate piroplasm diversity in wildlife and the cattle population of the greater Kafue ecosystem, we utilized PCR to amplify the 18S rRNA V4 hyper-variable region and meta-barcoding strategy using the Illumina MiSeq sequencing platform and amplicon sequence variant (ASV)-based bioinformatics pipeline to generate high-resolution data that discriminate sequences down to a single nucleotide difference. RESULTS: A parasite community of 45 ASVs corresponding to 23 species consisting of 4 genera of Babesia, Theileria, Hepatozoon and Colpodella, were identified in wildlife and the cattle population from the study area. Theileria species were detected in buffalo, impala, hartebeest, sable antelope, sitatunga, wild dog and cattle. In contrast, Babesia species were only observed in cattle and wild dog. Our results demonstrate possible spillover of these hemoprotozoan parasites from wildlife, especially buffalo, to the cattle population in the wildlife-livestock interface. CONCLUSION: We demonstrated that the deep amplicon sequencing of the 18S rRNA V4 hyper-variable region for wildlife was informative. Our results illustrated the diversity of piroplasma and the specificity of their hosts. They led us to speculate a possible ecological cycle including transmission from wildlife to domestic animals in the greater Kafue ecosystem. Thus, this approach may contribute to the establishment of appropriate disease control strategies in wildlife-livestock interface areas.

Development of a Multiplex Loop-Mediated Isothermal Amplification (LAMP) Method for Simultaneous Detection of Spotted Fever Group Rickettsiae and Malaria Parasites by Dipstick DNA Chromatography.

Spotted fever group (SFG) rickettsiae causes febrile illness in humans worldwide. Since SFG rickettsiosis's clinical presentation is nonspecific, it is frequently misdiagnosed as other febrile diseases, especially malaria, and complicates proper treatment. Aiming at rapid, simple, and simultaneous detection of SFG Rickettsia spp. and Plasmodium spp., we developed a novel multiple pathogen detection system by combining a loop-mediated isothermal amplification (LAMP) method and dipstick DNA chromatography technology. Two primer sets detecting SFG Rickettsia spp. and Plasmodium spp. were mixed, and amplified products were visualized by hybridizing to dipstick DNA chromatography. The multiplex LAMP with dipstick DNA chromatography distinguished amplified Rickettsia and Plasmodium targeted genes simultaneously. The determined sensitivity using synthetic nucleotides was 1000 copies per reaction for mixed Rickettsia and Plasmodium genes. When genomic DNA from in vitro cultured organisms was used, the sensitivity was 100 and 10 genome equivalents per reaction for Rickettsia monacensis and Plasmodium falciparum, respectively. Although further improvement will be required for more sensitive detection, our developed simultaneous diagnosis technique will contribute to the differential diagnosis of undifferentiated febrile illness caused by either SFG Rickettsia spp. or Plasmodium spp. in resource-limited endemic areas. Importantly, this scheme is potentially versatile for the simultaneous detection of diverse infectious diseases.

Development of a bio-inkjet printed LAMP test kit for detecting human African trypanosomiasis.

Human African trypanosomiasis (HAT) is one of the neglected tropical diseases in sub-Saharan Africa. Early diagnosis and treatment prior to disease progression are crucial for the survival of HAT patients. We had previously established a loop-mediated isothermal amplification (LAMP) method for HAT diagnosis in which the reagents were dried for field-use purposes. In this study, we used a semi-automated process to produce the test tubes using a bio-inkjet printer to achieve an accurate production. The performance of the inkjet printer-produced dried LAMP test (CZC-LAMP) was found to be stable after storage for up to 180 days at 30 °C. The diagnostic accuracy of CZC-LAMP HAT was evaluated using DNA samples that were extracted from 116 Trypanosoma brucei gambiense patients and 66 T. b. rhodesiense patients. The sensitivity was 72% for T. b. gambiense (95%CI: 63%-80%) and 80% for T. b. rhodesiense (95%CI: 69%-89%). The specificity determined using DNA from 116 endemic control DNA samples was 95% (95%CI: 89%-98%). The performance of the CZC-LAMP HAT and CZC-LAMP rHAT were also evaluated using 14 crude blood lysate samples obtained from T. b. rhodesiense patients and endemic control samples collected from Rumphi District in Malawi. The sensitivity and specificity were both 100% (95%CI: 77%-100%). As the developed CZC-LAMP test does not require a cold chain or a sophisticated laboratory, it holds promise for use as a routine simple molecular tool for point-of-care HAT diagnosis in endemic areas.

Molecular characterization of Cryptosporidium spp. from patients with diarrhoea in Lusaka, Zambia.

Cryptosporidium is a major etiological agent of diarrhoeal diseases among children and immune-compromised individuals in sub-Saharan African countries. We conducted a study to determine the prevalence and genetic characteristics of Cryptosporidium spp. in stool samples from patients with diarrhoea who presented at the University Teaching Hospital in Lusaka, Zambia. Cryptosporidium species and subtypes from 71 microscopically confirmed cryptosporidiosis stool samples collected between 2017 and 2019 were determined by polymerase chain reaction followed by partial sequencing of the small subunit rRNA and 60-kDa glycoprotein (gp60) gene. Additionally, data for the period between 2014 and 2019 were reviewed and analysed for cryptosporidiosis seasonal and age distribution. Cryptosporidium was more prevalent in the rainy season. The highest number of cases was reported among the 1-4 year age group. By sequence analysis of the 71 positive isolates, Cryptosporidium hominis (n = 42; 59.2%), C. parvum (n = 27; 38%), C. felis (n = 1; 1.4%), and C. meleagridis (n = 1; 1.4%) were identified. Four C. hominis subtype families (Ia, Ib, Id, and Ie) and three C. parvum subtype families (IIc, IIe, and IIs) were identified. The most frequent subtypes were IeA11G3T3 (n = 20; 28.2%), IIcA5G3 (n = 12; 16.9%), IIeA12G1 (n = 11; 15.5%) and IaA30R3 (n = 10; 14.1%). The observed species/subtypes of C. hominis and C. parvum indicated that the infection was mainly transmitted through the anthroponotic route. The identification of C. felis and C. meleagridis suggests that an atypical zoonotic transmission cycle also exists.

TITLE: Caractérisation moléculaire de Cryptosporidium spp. de patients souffrant de diarrhée à Lusaka, Zambie. ABSTRACT: Cryptosporidium est un agent étiologique majeur des maladies diarrhéiques chez les enfants et les personnes immunodéprimées dans les pays d'Afrique subsaharienne. Nous avons mené une étude pour déterminer la prévalence et les caractéristiques génétiques de Cryptosporidium spp. dans des échantillons de selles de patients souffrant de diarrhée qui se sont présentés à l'hôpital universitaire de Lusaka, en Zambie. Les espèces et sous-types de Cryptosporidium provenant de 71 échantillons de selles de cryptosporidiose, confirmés au microscope et prélevés entre 2017 et 2019, ont été déterminés par réaction en chaîne par polymérase suivie d'un séquençage partiel de la petite sous-unité de l'ARNr et du gène de la glycoprotéine de 60 kDa (gp60). De plus, les données pour la période entre 2014 et 2019 ont été examinées et analysées pour la distribution saisonnière et par âge de la cryptosporidiose. Cryptosporidium était plus répandu pendant la saison des pluies. Le plus grand nombre de cas a été signalé dans le groupe d'âge de 1 à 4 ans. Par analyse séquentielle des 71 isolats positifs, Cryptosporidium hominis (n = 42 ; 59,2 %), C. parvum (n = 27 ; 38 %), C. felis (n = 1 ; 1,4 %) et C. meleagridis (n = 1 ; 1,4 %) ont été identifiés. Quatre familles de sous-types de C. hominis (Ia, Ib, Id et Ie) et trois familles de sous-types de C. parvum (IIc, IIe et IIs) ont été identifiées. Les sous-types les plus fréquents étaient A11G3T3 (n = 20 ; 28,2 %), IIcA5G3 (n = 12 ; 16,9 %), IIeA12G1 (n = 11 ; 15,5 %) et IaA30R3 (n = 10 ; 14,1 %). Les espèces/sous-types observés de C. hominis et C. parvum indiquent que l'infection est principalement transmise par voie anthroponotique. L'identification de C. felis et C. meleagridis suggère qu'il existe également un cycle de transmission zoonotique atypique.

The Lethal(2)-Essential-for-Life [L(2)EFL] Gene Family Modulates Dengue Virus Infection in Aedes aegypti.

Efforts to determine the mosquito genes that affect dengue virus replication have identified a number of candidates that positively or negatively modify amplification in the invertebrate host. We used deep sequencing to compare the differential transcript abundances in Aedes aegypti 14 days post dengue infection to those of uninfected A. aegypti. The gene lethal(2)-essential-for-life [l(2)efl], which encodes a member of the heat shock 20 protein (HSP20) family, was upregulated following dengue virus type 2 (DENV-2) infection in vivo. The transcripts of this gene did not exhibit differential accumulation in mosquitoes exposed to insecticides or pollutants. The induction and overexpression of l(2)efl gene products using poly(I:C) resulted in decreased DENV-2 replication in the cell line. In contrast, the RNAi-mediated suppression of l(2)efl gene products resulted in enhanced DENV-2 replication, but this enhancement occurred only if multiple l(2)efl genes were suppressed. l(2)efl homologs induce the phosphorylation of eukaryotic initiation factor 2α (eIF2α) in the fruit fly Drosophila melanogaster, and we confirmed this finding in the cell line. However, the mechanism by which l(2)efl phosphorylates eIF2α remains unclear. We conclude that l(2)efl encodes a potential anti-dengue protein in the vector mosquito.