Benchmarking CRISPR-BP34 for point-of-care melioidosis detection in low-income and middle-income countries: a molecular diagnostics study.

BACKGROUND: Melioidosis is a neglected but often fatal tropical disease. The disease has broad clinical manifestations, which makes diagnosis challenging and time consuming. To improve diagnosis, we aimed to evaluate the performance of the CRISPR-Cas12a system (CRISPR-BP34) to detect Burkholderia pseudomallei DNA across clinical specimens from patients suspected to have melioidosis. METHODS: We conducted a prospective, observational cohort study of adult patients (aged ≥18 years) with melioidosis at Sunpasitthiprasong Hospital, a tertiary care hospital in Thailand. Participants were eligible for inclusion if they had culture-confirmed B pseudomallei infection from any clinical samples. Data were collected from patient clinical records and follow-up telephone calls. Routine clinical samples (blood, urine, respiratory secretion, pus, and other body fluids) were collected for culture. We documented time taken for diagnosis, and mortality at day 28 of follow-up. We also performed CRISPR-BP34 detection on clinical specimens collected from 330 patients with suspected melioidosis and compared its performance with the current gold-standard culture-based method. Discordant results were validated by three independent qualitative PCR tests. This study is registered with the Thai Clinical Trial Registry, TCTR20190322003. FINDINGS: Between Oct 1, 2019, and Dec 31, 2022, 876 patients with culture-confirmed melioidosis were admitted or referred to Sunpasitthiprasong Hospital, 433 of whom were alive at diagnosis and were enrolled in this study. Median time from sample collection to diagnosis by culture was 4·0 days (IQR 3·0-5·0) among all patients with known survival status at day 28, which resulted in delayed treatment. 199 (23%) of 876 patients died before diagnosis and 114 (26%) of 433 patients in follow-up were treated, but died within 28 days of admission. To test the CRISPR-BP34 assay, we enrolled and collected clinical samples from 114 patients with melioidosis and 216 patients without melioidosis between May 26 and Dec 31, 2022. Application of CRISPR-BP34 reduced the median sample-to-diagnosis time to 1·1 days (IQR 0·7-1·5) for blood samples, 2·3 h (IQR 2·3-2·4) for urine, and 3·3 h (3·1-3·4) for respiratory secretion, pus, and other body fluids. The overall sensitivity of CRISPR-BP34 was 93·0% (106 of 114 samples [95% CI 86·6-96·9]) compared with 66·7% (76 of 114 samples [57·2-75·2]) for culture. The overall specificity of CRISPR-BP34 was 96·8% (209 of 216 samples [95% CI 93·4-98·7]), compared with 100% (216 of 216 samples [98·3-100·0]) for culture. INTERPRETATION: The sensitivity, specificity, speed, and window of clinical intervention offered by CRISPR-BP34 support its prospective use as a point-of-care diagnostic tool for melioidosis. Future development should be focused on scalability and cost reduction. FUNDING: Chiang Mai University Thailand and Wellcome Trust UK.

Under-Reporting Cases and Deaths from Melioidosis: A Retrospective Finding in Songkhla and Phatthalung Province of Southern Thailand, 2014-2020.

Melioidosis, caused by Burkholderia pseudomallei, is a notifiable disease associated with a high mortality rate in Thailand. The disease is highly endemic in northeast Thailand, while its prevalence in other parts of the country is poorly documented. This study aimed at improving the surveillance system for melioidosis in southern Thailand, where the disease was believed to be underreported. Two adjacent southern provinces, Songkhla and Phatthalung, were selected as the model provinces to study melioidosis. There were 473 individuals diagnosed with culture-confirmed melioidosis by clinical microbiology laboratories at four tertiary care hospitals in both provinces from January 2014 to December 2020. The median age was 54 years (IQR 41.5-64), 284 (60%) of the patients were adults ≥50 years of age, and 337 (71.2%) were male. We retrospectively analyzed 455 patients treated at either Songklanarind Hospital, Hatyai Hospital, Songkhla Provincial Hospital, or Phatthalung Provincial Hospital, of whom 181 (39.8%) patients died. The median duration from admission to death was five days (IQR 2-17). Of the 455 patients, 272 (57.5%) had at least one clinical risk factor, and 188 (39.8%) had diabetes. Two major clinical manifestations, bacteremia and pneumonia, occurred in 274 (58.1%) and 166 (35.2%) patients, respectively. In most cases, 298 (75%) out of 395 local patients were associated with rainfall. Over the seven years of the study, the average annual incidence was 2.87 cases per 100,000 population (95% CI, 2.10 to 3.64). This study has confirmed that these two provinces of southern Thailand are endemic to melioidosis; even though the incidence rate is much lower than that of the Northeast, the mortality rate is comparably high.

Alteration of monocyte subsets and their functions in thalassemia patients.

Infection is one of the leading causes of mortality in thalassemia patients. This study aimed to examine qualitative and quantitative changes in monocytes in thalassemia patients. Monocytes were isolated from peripheral blood mononuclear cells and separated into subpopulations by flow cytometry. Cytokine levels were measured using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and sandwich enzyme-linked immunosorbent assay (ELISA). The primary endpoint was monocyte-derived TNF-α expression. A total of 78 patients and 26 controls were included. The mean log (TNF-α fold-change) by qRT-PCR was significantly lower in all thalassemia groups, at 1.27 in controls, versus 0.97 (p = 0.0014) in non-transfusion-dependent thalassemia (NTDT), 0.96 (p = 0.0004) in non-splenectomized transfusion-dependent thalassemia (TDT-NS), and 0.87 (p < 0.0001) in splenectomized transfusion-dependent thalassemia (TDT-S). Similarly, the mean 2-h TNF-α level measured by sandwich ELISA assay was significantly lower in all thalassemia groups, at 98.16 pg/mL in controls, versus 56.45 pg/mL (p = 0.0093) in NTDT, 39.05 pg/mL (p = 0.0001) in TDT-NS and 32.37 pg/mL (p < 0.0001) in TDT-S. Likewise, TDT patients had a significantly decreased percentage of non-classical monocytes, by approximately half compared to controls. Our results show that thalassemia major patients have clearly impaired monocyte counts and function.

Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a.

Detection of Burkholderia pseudomallei, a causative bacterium for melioidosis, remains a challenging undertaking due to long assay time, laboratory requirements, and the lack of specificity and sensitivity of many current assays. In this study, we are presenting a novel method that circumvents those issues by utilizing CRISPR-Cas12a coupled with isothermal amplification to identify B. pseudomallei DNA from clinical isolates. Through in silico search for conserved CRISPR-Cas12a target sites, we engineered the CRISPR-Cas12a to contain a highly specific spacer to B. pseudomallei, named crBP34. The crBP34-based detection assay can detect as few as 40 copies of B. pseudomallei genomic DNA while discriminating against other tested common pathogens. When coupled with a lateral flow dipstick, the assay readout can be simply performed without the loss of sensitivity and does not require expensive equipment. This crBP34-based detection assay provides high sensitivity, specificity and simple detection method for B. pseudomallei DNA. Direct use of this assay on clinical samples may require further optimization as these samples are complexed with high level of human DNA.

Attempt to Isolate Elephant Endotheliotropic Herpesvirus (EEHV) Using a Continuous Cell Culture System.

Elephant endotheliotropic herpesvirus (EEHV) infection is known to cause acute fatal hemorrhagic disease, which has killed many young Asian elephants (Elephas maximus). Until recently, in vitro isolation and propagation of the virus have not been successful. This study aimed to isolate and propagate EEHV using continuous cell lines derived from human and/or animal origins. Human cell lines, including EA. hy926, A549, U937, RKO, SW620, HCT-116 and HT-29, and animal cell lines, including CT26.CL25 and sp2/0-Ag14, were investigated in this study. Mixed frozen tissue samples of the heart, lung, liver, spleen and kidney obtained from fatal EEHV1A- or EEHV4-infected cases were homogenized and used for cell inoculation. At 6, 24, 48 and 72 h post infection (hpi), EEHV-inoculated cells were observed for cytopathic effects (CPEs) or were assessed for EEHV infection by immunoperoxidase monolayer assay (IPMA) or quantitative PCR. The results were then compared to those of the mock-infected controls. Replication of EEHV in the tested cells was further determined by immunohistochemistry of cell pellets using anti-EEHV DNA polymerase antibodies or re-inoculated cells with supernatants obtained from passages 2 or 3 of the culture medium. The results reveal that no CPEs were observed in the tested cells, while immunolabeling for EEHV gB was observed in only U937 human myeloid leukemia cells. However, quantitation values of the EEHV terminase gene, as well as those of the EEHV gB or EEHV DNA polymerase proteins in U937 cells, gradually declined from passage 1 to passage 3. The findings of this study indicate that despite poor adaptation in U937 cells, this cell line displays promise and potential to be used for the isolation of EEHV1 and EEHV4 in vitro.

A Cost Effective Easy Competitive Enzyme-Linked Immunosorbent Assay Suitable for Monitoring Protective Immunity against the Rabies Virus in the Serum of Humans and Dogs.

The coverage of rabies vaccinations has been reported at 70-80% of dogs in annual reports. However, there are still outbreaks of rabies among humans and dogs in Thailand, thus indicating the necessity of ensuring seroprevalence in vaccinated dogs and efficacy of human immunization. A cost effective easy competitive enzyme-linked immunosorbent assay (CEE-cELISA) was developed here for monitoring protective immunity against the rabies virus in human and dog serum samples using monoclonal antibody clone 1-46-12, which recognizes a conformational epitope of the rabies G protein. The ELISA plate is coated with the whole viral antigen from a commercial vaccine. The serotiter measured by the CEE-cELISA and by the gold standard assay (rapid fluorescent focus inhibition test), detecting the neutralizing antibody, showed a strong correlation, with an R value of 0.958 and 0.931 in humans and dogs, respectively. These correlations were detected in the serum samples from humans and dogs at antibody concentrations up to 100 and 10 IU/ml, respectively. This CEE-cELISA could be an alternative assay for evaluating mass rabies vaccination rapidly at a low cost as well as for detecting antirabies antibodies in the serum of not only humans but also other animal species.

Hepatoprotective effects of lychee (Litchi chinensis Sonn.): a combination of antioxidant and anti-apoptotic activities.

AIM OF THE STUDY: Gimjeng and Chakapat lychee (Litchi chinensis Sonn.) were evaluated for hepatoprotective activity on CCl(4)-induced hepatotoxicity in rats. MATERIALS AND METHODS: Fruit pulp extracts of the lychees were examined for vitamin C, phenolic contents, anti-lipid peroxidation activity and hepatoprotective effect. Male Wistar albino rats were intraperitoneally injected (ip) with CCl(4) (2 ml/kg), then were orally administered (po) with silymarin (100mg/kg), and Gimjeng or Chakapat extracts (100 and 500 mg/kg). After ten days, the rats were sacrificed and their livers were examined histopathologically and immunohistochemically. Their serum glutamate-pyruvate transaminase, glutamate-oxalate transaminase, and alkaline phosphatase activities were analyzed. Apoptotic activity of the livers was assessed quantitatively. RESULTS: The Gimjeng and Chakapat extracts showed the contents of vitamin C (1.2±0.6 and 4.3±0.1mg/100g) and phenolics like trans-cinnamic acid and pelargonidin-3-O-glucoside (9.80±0.21 and 19.56±0.4 mg GAE/g extract, respectively), and trolox equivalent antioxidant capacity (TEAC) values (11.64 and 9.09 g/mg trolox), respectively. The Gimjeng as compared to the Chakapat demonstrated a better antioxidant activity as revealed by anti-lipid peroxidation activity with the TEAC values. Administration of CCl(4) in rats elevated the serum GPT, GOT, and ALP level whereas silymarin, Gimjeng and Chakapat extracts prevented these increases significantly. Significant decrease of apoptotic cells together with restoration of morphological changes confirmed the hepatoprotective effect in the CCl(4)-induced rats pretreated with the extracts. CONCLUSION: Antioxidant properties of the Gimjeng and Chakapat lychees as evidenced by the vitamin C and phenolic compounds, anti-lipid peroxidation and anti-apoptosis could explain the hepatoprotective effects in CCl(4)-induced hepatotoxicity.

Dimorphic motifs in D0 and D1+D2 domains of killer cell Ig-like receptor 3DL1 combine to form receptors with high, moderate, and no avidity for the complex of a peptide derived from HIV and HLA-A*2402.

Comparison of mutant killer cell Ig-like receptor (KIR) 3DL1*015 substituted at natural positions of variation showed that tryptophan/leucine dimorphism at position 283 uniquely changes receptor conformation and can strongly influence binding of the A24nef tetramer. Dimorphic motifs at positions 2, 47, and 54 in D0 and 182 and 283 in D1+D2 distinguish the two 3DL1 lineages, typified by 3DL1*005 and 3DL1*015. The interlineage recombinant, KIR3DL1*001, combines D0 of 3DL1*005 with D1+D2 of 3DL1*015 and binds A24nef more strongly than either parent. In contrast, the reciprocal recombinant with D0 from 3DL1*015 and D1+D2 from 3DL1*005 cannot bind A24nef. Thus, D0 polymorphism directly affects the avidity of the KIR3DL1 ligand binding site. From these observations, multiple sequence alignment, and homology modeling, we constructed structural models for KIR3DL1 and its complex with A24nef. In these models, D0, D1, and D2 come together to form a binding surface for A24nef, which is contacted by all three Ig-like domains. A central pocket binds arginine 83, the only Bw4 motif residue essential for KIR3DL1 interaction, similar to the binding of lysine 80 in HLA-C by KIR2DL1. Central to this interaction is a salt bridge between arginine 83 of Bw4 and glutamate 282 of 3DL1, which juxtaposes the functionally influential dimorphism at position 283. Further 3DL1 mutants were tested and shown to have A24nef-binding properties consistent with the models. A24nef was not bound by KIR3DS1, the activating counterpart of KIR3DL1. Moreover, introducing any one of three residues specific to KIR3DS1, serine 163, arginine 166, or leucine 199, into 3DL1*015, abrogated A24nef binding.

Reciprocal recognition of an HLA-Cw4-restricted HIV-1 gp120 epitope by CD8+ T cells and NK cells.

OBJECTIVES: The HIV-1 Nef protein selectively downregulates human leukocyte antigen (HLA)-A and HLA-B but not HLA-C molecules on the surface of infected cells. This allows HIV-infected cells to evade recognition by most cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. We investigated the recognition of an HLA-Cw4-restricted HIV-1 gp120 epitope SFNCGGEFF (SF9) and its variant SFNCGGEFL (SL9) by T cells and NK receptors. DESIGN AND METHOD: Recognition of HIV-1 gp120 peptides (SF9 and SL9) by T-cell clones was measured by staining with HLA-Cw4-peptide tetrameric complexes and cytolytic assays using target cell pulsed with either peptides. KIR2DL1 binding to these two peptides was measured using surface plasmon resonance and tetramer staining of an NK cell line. RESULT: : CTLs could recognize SF9 better than the variant SL9, as shown by both tetramer staining and cytolytic assays. Intriguingly, an HLA-Cw4 tetramer folded with the 'escape' variant SL9 could bind to KIR2DL1 on NK cell lines with higher affinity than HLA-Cw4-SF9. The binding of KIR2DL1 to its ligand results in inhibition of NK cell function. Our results indicate that the HIV-1 gp120 variant peptide SL9 could potentially escape both from NK cell and CTL recognition by increasing its affinity for KIR2DL1 binding. CONCLUSION: These data suggest that HIV-1 can acquire mutations that are capable of escaping from both CTL and NK cell recognition, a phenomenon we have termed 'double escape'.

Cutting Edge: Allele-specific and peptide-dependent interactions between KIR3DL1 and HLA-A and HLA-B.

Although it is clear that KIR3DL1 recognizes Bw4(+) HLA-B, the role of Bw4(+) HLA-A allotypes as KIR3DL1 ligands is controversial. We therefore examined the binding of tetrameric HLA-A and -B complexes, including HLA*2402, a common Bw4(+) HLA-A allotype, to KIR3DL1*001, *005, *007, and *1502 allotypes. Only Bw4(+) tetramers bound KIR3DL1. Three of four HLA-A*2402 tetramers bound one or more KIR3DL1 allotypes and all four KIR3DL1 allotypes bound to one or more HLA-A*2402 tetramers, but with different binding specificities. Only KIR3DL1*005 bound both HLA-A*2402 and HLA-B*5703 tetramers. HLA-A*2402-expressing target cells were resistant to lysis by NK cells expressing KIR3DL1*001 or *005. This study shows that HLA-A*2402 is a ligand for KIR3DL1 and demonstrates how the binding of KIR3DL1 to Bw4(+) ligands depends upon the bound peptide as well as HLA and KIR3DL1 polymorphism.

Recognition of HLA-A3 and HLA-A11 by KIR3DL2 is peptide-specific.

The recognition of MHC class I molecules by killer cell immunoglobulin-like receptors (KIR) is central to the control of NK cell function and can also modulate the CTL activation threshold. Among KIR receptors, KIR3DL2 is thought to interact with HLA-A3 and -A11, although direct evidence has been lacking. In this study, we show that HLA-A3 and -A11 tetramers specifically bind to KIR3DL2*001 transfectants and that this recognition is peptide-specific. Single amino acid substitutions in the nonamer peptide underline a critical role for residue 8 in recognition of KIR3DL2. However, the role of this interaction in vivo still remains to be established.