Development of a Simple Method to Detect the Carbapenemase-Producing Genes blaNDM, blaOXA-48-like, blaIMP, blaKPC, and blaVIM Using a LAMP Method with Lateral Flow DNA Chromatography.

Infections by carbapenemase-producing Enterobacterales constitute a global public health threat. The rapid and efficient diagnosis of Enterobacterales infection is critical for prompt treatment and infection control, especially in hospital settings. We developed a novel loop-mediated isothermal amplification (LAMP) method combined with DNA chromatography to identify five major groups of carbapenemase-producing genes (blaNDM, blaOXA-48-like, blaIMP, blaKPC, and blaVIM). This method uses DNA-DNA hybridization-based detection in which LAMP products can be easily visualized as colored lines. No specific technical expertise, expensive equipment, or special facilities are required for this method, allowing its broad application. Here, 73 bacteria collections including strains with carbapenemase-producing genes were tested. Compared to sequencing results, LAMP DNA chromatography for five carbapenemase-producing genes had a sensitivity and specificity of 100% and >97%, respectively. This newly developed method can be a valuable rapid diagnostic test to guide appropriate treatments and infection control measures, especially in resource-limited settings.

Minimum inhibitory concentrations of azithromycin in clinical isolates of Mycobacterium avium complex in Japan.

The latest guidelines include azithromycin as a preferred regimen for treating Mycobacterium avium complex (MAC) pulmonary disease. However, serially collected susceptibility data on clinical MAC isolates are limited, and no breakpoints have been determined. We investigated the minimum inhibitory concentrations (MICs) of azithromycin and clarithromycin for all MAC strains isolated in 2021 from a single center in Japan, excluding duplicates. The MICs were determined using a panel based on the microbroth dilution method, according to the latest Clinical and Laboratory Standards Institute recommendations. The MICs were determined for 318 MAC strains. Although there was a significant positive correlation between the MICs of azithromycin and clarithromycin, the MICs of azithromycin tended to be higher than those of clarithromycin. Among the cases in which the strains were isolated, 18 patients initiated treatment, including azithromycin treatment, after sample collection. Some patients infected with stains with relatively high azithromycin MICs achieved a microbiological cure with azithromycin-containing regimens. This study revealed a higher MIC distribution for azithromycin than clarithromycin, raising questions about the current practice of estimating azithromycin susceptibility based on the clarithromycin susceptibility test result. However, this was a single-center study that included only a limited number of cases treated with azithromycin. Therefore, further multicenter studies that include a greater number of cases treated with azithromycin are warranted to verify the distribution of azithromycin MICs and examine the correlation between azithromycin MICs and treatment effectiveness.IMPORTANCEThe macrolides serve as key drugs in the treatment of pulmonary Mycobacterium avium complex infection, and the administration of macrolide should be guided by susceptibility test results. Azithromycin is recommended as a preferred choice among macrolides, surpassing clarithromycin; however, drug susceptibility testing is often not conducted, and clarithromycin susceptibility is used as a surrogate. This study represents the first investigation into the minimum inhibitory concentration of azithromycin on a scale of several hundred clinical isolates, revealing an overall tendency for higher minimum inhibitory concentrations compared with clarithromycin. The results raise questions about the appropriateness of using clarithromycin susceptibility test outcomes for determining the administration of azithromycin. This study highlights the need for future discussions on the clinical breakpoints of azithromycin, based on large-scale clinical research correlating azithromycin susceptibility with treatment outcomes.

Cost-Effectiveness of Universal Asymptomatic Preoperative SARS-CoV-2 Polymerase Chain Reaction Screening: A Cost-Utility Analysis.

BACKGROUND: An early report has shown the clinical benefit of the asymptomatic preoperative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) screening test, and some clinical guidelines recommended this test. However, the cost-effectiveness of asymptomatic screening was not evaluated. We aimed to investigate the cost-effectiveness of universal preoperative screening of asymptomatic patients for SARS-CoV-2 using polymerase chain reaction (PCR) testing. METHODS: We evaluated the cost-effectiveness of asymptomatic screening using a decision tree model from a payer perspective, assuming that the test-positive rate was 0.07% and the screening cost was 8500 Japanese yen (JPY) (approximately 7601 US dollars [USD]). The input parameter was derived from the available evidence reported in the literature. A willingness-to-pay threshold was set at 5 000 000 JPY/quality-adjusted life-year (QALY). RESULTS: The incremental cost of 1 death averted was 74 469 236 JPY (approximately 566 048 USD) and 291 123 368 JPY/QALY (approximately 2 212 856 USD/QALY), which was above the 5 000 000 JPY/QALY willingness-to-pay threshold. The incremental cost-effectiveness ratio fell below 5 000 000 JPY/QALY only when the test-positive rate exceeded 0.739%. However, when the probability of developing a postoperative pulmonary complication among SARS-CoV-2-positive patients was below 0.22, asymptomatic screening was never cost-effective, regardless of how high the test-positive rate became. CONCLUSIONS: Asymptomatic preoperative universal SARS-CoV-2 PCR screening is not cost-effective in the base case analysis. The cost-effectiveness mainly depends on the test-positive rate, the frequency of postoperative pulmonary complications, and the screening costs; however, no matter how high the test-positive rate, the cost-effectiveness is poor if the probability of developing postoperative pulmonary complications among patients positive for SARS-CoV-2 is sufficiently reduced.

[Cerebral toxoplasmosis developed after unrelated bone marrow transplantation for acute myeloid leukemia].

A 16-year-old boy received an unrelated bone marrow transplant while in second remission of acute myeloid leukemia. He suffered from severe oral mucosal complications and had difficulty taking oral drugs such as sulfamethoxazole/trimethoprim (ST). Engraftment was obtained on transplant day 35, and blurred vision and headache appeared around transplant day 60. Funduscopy revealed retinal hemorrhage and macular edema, and an MRI scan of the head revealed a nodular lesion in the left putamen. Toxoplasma gondii was detected by CSF PCR, and cerebral toxoplasmosis was diagnosed. Following therapy with ST and clindamycin, the patient was administered pyrimethamine, sulfadiazine, and leucovorin. Symptoms improved promptly, and CSF PCR was negative 45 days after the start of treatment. Since the prevalence of toxoplasma antibodies increases with age, it is crucial to avoid toxoplasma reactivation by ST after hematopoietic cell transplantation in postpubescent patients.

Disseminated Toxoplasmosis associated with Haemophagocytic Lymphohistiocytosis in a Patient with the Human Immunodeficiency Virus: A Case Report and Literature Review.

Disseminated toxoplasmosis associated with haemophagocytic lymphohistiocytosis (DT-HLH) is rare and difficult to diagnose compared to disseminated toxoplasmosis or HLH presenting alone. Because of the limited number of reported cases, the clinical characteristics and outcomes of DT-HLH are unknown. We report a case of DT-HLH in a human immunodeficiency virus (HIV)-infected patient who was successfully treated with early anti-toxoplasmic therapy and performed a comprehensive literature review. A 33-year-old Cameroonian woman was transferred to our hospital owing to HIV infection and encephalitis. Although she developed HLH, bone marrow biopsy did not reveal the cause. She was diagnosed as having DT-HLH via polymerase chain reaction testing of bone marrow biopsy tissue, blood, and cerebrospinal fluid. DT-HLH improved within the initial two weeks of treatment for toxoplasmosis (sulfamethoxazole-trimethoprim, trimethoprim 10 mg/kg/day and clindamycin 1,800 mg/day) before the introduction of antiretroviral therapy. To our knowledge, only eight cases of DT-HLH have been previously reported in the literature. Most patients died within three weeks of hospitalisation and were diagnosed by autopsy. Conversely, patients diagnosed antemortem were all treated and survived, including the currently reported patient. DT-HLH can lead to poor prognosis without early and proper treatment. Clinicians should consider toxoplasmosis in the differential diagnosis of HLH.

Cerebral toxoplasmosis with multiple hemorrhage lesions in an HIV infected patient: A case report and literature review.

A 47-year-old man was admitted to a hospital for disturbance of consciousness. He was diagnosed with multiple hemorrhagic brain abscesses in bilateral hemispheres with human immunodeficiency virus (HIV) infection, and was transferred to our hospital for further examination and treatment. On admission, although he could respond to pain stimuli, he could not talk or communicate. His laboratory data on admission revealed CD4-positive T cell count of 67 cells/µL, and HIV1-RNA viral load of 5.6 × 105 copies/mL. Both the serum IgG Toxoplasma gondii antibody and the cerebrospinal fluid polymerase chain reaction for Toxoplasma gondii DNA were positive. He was diagnosed with cerebral toxoplasmosis and HIV infection. His level of consciousness worsened, and the number of hemorrhagic lesions had increased in both hemispheres and the left thalamus on the computed tomography scan following two weeks of antitoxoplasma therapy. These newly discovered hemorrhagic lesions revealed in the CT had been found as the high intensity signal regions of initial fluid-attenuated inversion recovery magnetic resonance imaging. After five weeks of treatment, the hemorrhagic lesions gradually improved along with the patient's consciousness. Antiretroviral therapy was initiated six weeks following antitoxoplama therapy with reassurance that immune reconstitution inflammatory syndrome did not occur. After approximately four months of antitoxoplasma therapy, the patient was discharged into a group home with residual left hemiparesis on maintenance antitoxoplasma and antiretroviral therapy. Clinicians should recognize the delay of clinical and radiological improvement for hemorrhagic cerebral toxoplasmosis and patiently continue the antitoxoplasma therapy.

Toxoplasmosis after allogeneic hematopoietic stem cell transplantation: Impact of serostatus-based management.

Toxoplasmosis caused by Toxoplasma gondii (T. gondii) is a serious infectious complication after allogeneic hematopoietic stem cell transplantation (HSCT). The incidence of toxoplasmosis varies widely because of the variabilities of seroprevalence among patient populations. The incidence and the optimal management of toxoplasmosis after allogeneic HSCT in a patient population with a low seroprevalence have not been fully evaluated. We conducted a single-center retrospective study evaluating toxoplasmosis in Japanese patients who underwent allogeneic HSCT. Of the 728 evaluable patients, only 5 developed toxoplasmosis with a median onset of day 60 post-transplant (range, day 55-393). The cumulative incidence was 0.7% (95% CI: 0.3%-1.5%) at day 500 post-transplant. Four of the five patients succumbed due to toxoplasmosis. The more recently treated 220 patients (not the earlier 508 patients) were screened for the T. gondii serostatus, and prophylactic treatment with trimethoprim/sulfamethoxazole was applied. All five patients with toxoplasmosis were in the unscreened group, and there was no case of toxoplasmosis after the introduction of the screening and prophylactic treatment. Our results suggest that toxoplasmosis after allogeneic HST is rare but can develop as a life-threatening complication even in the populations with low seroprevalence, and that prophylactic treatment for seropositive patients could effectively prevent toxoplasmosis.

Chagas disease: a report of 17 suspected cases in Japan, 2012-2017.

BACKGROUND: There are no reports on the prevalence of Chagas disease in Japan. Furthermore, screening programs and access to diagnosis and treatment have not been established. This study aimed to clarify the prevalence of Chagas disease among suspected cases in Japan and provide the reference data required for disease control. METHODS: Seventeen patients with suspected Chagas disease in Japan between 2012 and 2017 were included in the study. Patients were diagnosed with Chagas disease based on the two different serological tests for antibodies to Trypanosoma cruzi. Real-time polymerase chain reaction assay and blood culture techniques were performed to confirm T. cruzi parasitemia. RESULTS: Of the 17 patients, 11 (64.7%) were immigrants from Latin America. Ultimately, 6 patients (35.3%) were diagnosed with Chagas disease. Of these 6 patients, median age was 53.5 years, 5 patients were immigrants from Latin American, and 1 was Japanese who had a congenital infection. T. cruzi parasitemia was confirmed in 4 patients (66.7%), and 5 (83.3%) were in the chronic phase (Chagas cardiomyopathy, 4; megacolon, 1). Two patients (33.3%) commenced benznidazole treatment. CONCLUSION: Our study showed that some patients of Chagas disease living in Japan are already in the chronic phase at diagnosis because of substantial diagnostic delays. Further epidemiological studies on the prevalence of Chagas disease and systematic screening programs for the Latin American population are needed.

Non-invasive diagnosis of cutaneous leishmaniasis by the direct boil loop-mediated isothermal amplification method and MinION™ nanopore sequencing.

Cutaneous leishmaniasis (CL) is gaining attention as a public health problem. We present two cases of CL imported from Syria and Venezuela in Japan. We diagnosed them as CL non-invasively by the direct boil loop-mediated isothermal amplification method and an innovative sequencing method using the MinION™ sequencer. This report demonstrates that our procedure could be useful for the diagnosis of CL in both clinical and epidemiological settings.

Development of a highly resolved loop-mediated isothermal amplification method to detect the N526K ftsI mutation of ß-lactamase-negative ampicillin-resistant Haemophilus influenzae.

Rapid and easy detection of sequence polymorphisms, including nucleotide point mutations of bacterial pathogens responsible for amino acid substitutions linked to drug resistance, is essential for the proper use of antimicrobial agents. Here, a detection method using loop-mediated amplification (LAMP) combined with amplification refractory mutation system (ARMS) to accurately distinguish a different single nucleotide in the target sequence was established, named ARMS-SNP LAMP. This procedure is capable of species-specific detection of a nucleotide (1578T) in the ftsI gene on Haemophilus influenzae without amplifying the sequence carrying the point mutations (T1578G/A) in ß-lactamase-negative ampicillin resistant (BLNAR) strains. Reactions were performed at 61°C for 45min. Successful target gene amplifications were detected by measuring real-time turbidity using a turbidimeter and visual detection. The assay had a detection limit of 10.0pg of genomic DNA per reaction and showed specificity against 52 types of pathogens, whereas amplifications were completely blocked in even 100.0ng/µL of genomic DNA with point mutations at T1578G and T1578A. The expected ARMS-SNP LAMP products were confirmed through identical melting curves in real-time LAMP procedures. This novel procedure was also used to analyze 57 clinical isolates of H. influenzae. All 25 clinical isolates with the naïve sequence of 1578T gave positive results. In addition, concordant negative results were obtained for 31 of the BLNAR strains with the T1578G mutation and one strain with the T1578A mutation. The ARMS-SNP LAMP method is a simple and rapid method for SNP-genotyping of a clinical isolate as point-of-care testing (POCT) technology. It is suitable for use in both resource-limited situations and well-equipped clinical settings because of its simplicity and convenience.

Point of care diagnosis of multiple schistosome parasites: Species-specific DNA detection in urine by loop-mediated isothermal amplification (LAMP).

Schistosomes are easily transmitted and high chance of repeat infection, so if control strategies based on targeted mass drug administration (MDA) are to succeed it is essential to have a test that is sensitive, accurate and simple to use. It is known and regularly demonstrated that praziquantel does not always eliminate an infection so in spite of the successes of control programs a residual of the reservoir survives to re-infect snails. The issue of diagnostic sensitivity becomes more critical in the assessment of program effectiveness. While serology, such as antigen capture tests might improve sensitivity, it has been shown that the presence of species-specific DNA fragments will indicate, most effectively, the presence of active parasites. Polymerase chain reaction (PCR) can amplify and detect DNA from urine residue captured on Whatman No. 3 filter paper that is dried after filtration. Previously we have detected S. mansoni and S. haematobium parasite-specific small repeat DNA fragment from filtered urine on filter paper by PCR. In the current study, we assessed the efficacy of detection of 86 urine samples for either or both schistosome parasites by PCR and loop-mediated isothermal amplification (LAMP) that were collected from a low to moderate transmission area in Ghana. Two different DNA extraction methods, standard extraction kit and field usable LAMP-PURE kit were also evaluated by PCR and LAMP amplification. With S. haematobium LAMP amplification for both extractions showed similar sensitivity and specificity when compared with PCR amplification (100%) verified by gel electrophoresis. For S. mansoni sensitivity was highest for LAMP amplification (100%) for standard extraction than PCR and LAMP with LAMP-PURE (99% and 94%). The LAMP-PURE extraction produced false negatives, which require further investigation for this field usable extraction kit. Overall high positive and negative predictive values (90% - 100%) for both species demonstrated a highly robust approach. The LAMP approach is close to point of care use and equally sensitive and specific to detection of species-specific DNA by PCR. LAMP can be an effective means to detect low intensity infection due to its simplicity and minimal DNA extraction requirement. This will enhance the effectiveness of surveillance and MDA control programs of schistosomiasis.

Progressive HIV-associated Cholangiopathy in an HIV Patient Treated with Combination Antiretroviral Therapy.

We herein describe a case of progressive human immunodeficiency virus (HIV)-associated cholangiopathy despite normalization of laboratory parameters, which had indicated liver dysfunction, after the initiation of combined anti-retroviral therapy (cART). HIV-associated cholangiopathy remains important as a differential diagnosis of bile duct disorders, although it is considered to be a rare disease in the era of cART. Magnetic resonance cholangiopancreatography could thus be a powerful tool for the diagnosis and follow-up of this disease.

Chikungunya Fever in Japan Imported from the Caribbean Islands.

A 53-year-old Japanese woman who was working as a volunteer in the Commonwealth of Dominica in the Caribbean islands presented with a high-grade fever and severe incapacitating generalized arthralgia. The Asian genotype of the chikungunya virus was confirmed using reverse transcription-PCR and serology, based on the presence of a specific neutralization titer and immunoglobulin M antibodies. She was diagnosed with post-chikungunya chronic arthritis based on persistence of her polyarthritis for 3 months and the presence of rheumatoid factor, immunoglobulin G-rheumatoid factor, and matrix metalloproteinase-3. Chikungunya virus should be considered as a causative pathogen in travelers returning from Caribbean islands. Clinicians should consider chikungunya fever in the differential diagnosis of patients who complain of chronic arthritis and have a history of travel to an endemic area.

PURE-LAMP Procedure for the Diagnosis of Extrapulmonary Tuberculosis: A Case Series.

Although the polymerase chain reaction is effective for the diagnosis of extrapulmonary tuberculosis (EPTB), it is typically unavailable in resource-limited situations. In contrast, the loop-mediated isothermal amplification (LAMP) assay is a relatively cost-effective and accessible method. Additionally, when combined with the procedure for ultra-rapid extraction (PURE) kit, which enables simple DNA extraction, LAMP can detect Mycobacterium tuberculosis in sputum within 1.5 hours using a simple procedure. In this study, we investigated the utility of the PURE-LAMP technique to diagnose three cases of EPTB and showed that it may potentially be a valuable tool for the diagnosis of EPTB.

Chronic Chagas disease with advanced cardiac complications in Japan: Case report and literature review.

Due to the unprecedented recent increases in global migration, Chagas disease has become a global health threat and its epidemiology has drastically changed. Here we describe the first case in Japan of benznidazole treatment for chronic Chagas disease characterized by advanced cardiac complications. A 55-year-old Japanese-Brazilian woman who had previously presented with chronic heart failure was diagnosed as having Chagas disease and treated with benznidazole to prevent aggravation of her cardiac complications. However, benznidazole administration was stopped on day 56 due to severe drug-induced peripheral neuritis. Sixteen months later, her serologic test for Trypanosoma cruzi is still positive and she is being followed regularly by cardiology. Despite an estimated prevalence of over 4000 cases in Japan, only a few cases of Chagas disease have been reported. A Medline search revealed only 7 cases identified between 1995 and 2014 in Japan: in 6 cases, complications of chronic Chagas disease were apparent at the time of presentation, and sudden death occurred in 2 of these cases due to cardiac complications. This clinical case and literature review re-emphasize the urgent need to establish a surveillance network and improve the diagnostic methods and treatment framework for Chagas disease in Japan.

Loop-mediated isothermal amplification with the Procedure for Ultra Rapid Extraction kit for the diagnosis of pneumocystis pneumonia.

Loop-mediated isothermal amplification (LAMP) is an innovative molecular technique requiring only a heating device and isothermal conditions to amplify a specific target gene. The results of current microscopic diagnostic tools for pneumocystis pneumonia are not sufficiently consistent for detecting infection with a low-density of Pneumocystis jirovecii. Although polymerase chain reaction (PCR) is highly sensitive, it is not suitable for resource-limited facilities. LAMP is a potential diagnostic replacement for PCR in such settings but a critical disadvantage of DNA extraction was still remained. Therefore, we employed the Procedure for Ultra Rapid Extraction (PURE) kit, which uses a porous material, to isolate the DNA from clinical samples in a simple way in combination with previously reported LAMP procedure for diagnosing PCP. The detection limit of the PURE-LAMP method applied to artificial bronchoalveolar lavage fluid samples was 100 copies/tube, even with the use of massive blood-contaminated solutions. In addition, we concluded the diagnostic procedure within 1 h without the need for additional equipment. PURE-LAMP coupled with suitable primers for specific pathogens has good potential for diagnosing various infectious diseases.

Human herpes virus-8-associated multicentric Castleman's disease in an HIV-positive patient presenting with relapsing and remitting hyponatraemia.

We report a case of human herpes virus-8-associated multicentric Castleman's disease in an HIV-positive patient with hyponatraemia. A 65-year-old man was admitted with relapsing and remitting fever, scattered skin eruptions and hepatosplenomegaly following combination antiretroviral therapy for his HIV infection. Based on histopathological findings, he was diagnosed as having human herpes virus-8-associated multicentric Castleman's disease and was treated with four-weekly infusions of rituximab. Prior to receiving chemotherapy, we observed several suspected biomarkers of disease activity, positive correlations between plasma human herpes virus-8 viral load and the levels of plasma interleukin-6, C-reactive protein and soluble interleukin-2 receptor, and negative correlations between platelet count, albumin levels and especially serum sodium levels. We hypothesize that non-osmotic release of plasma antidiuretic hormone is a cause of hyponatraemia in human herpes virus-8-associated multicentric Castleman's disease and that relapsing and remitting hyponatraemia could be correlated with plasma human herpes virus-8 viral load.

The Direct Boil-LAMP method: a simple and rapid diagnostic method for cutaneous leishmaniasis.

Needle biopsy is widely used for the diagnosis of cutaneous leishmaniasis (CL) to obtain specimens for histology and culture. However, the use of such invasive procedures causes discomfort, requires technical expertise, and carries risks of bleeding and iatrogenic infection. Therefore, developing substitutive non-invasive diagnostic tools for CL will help reduce the risk of secondary infection and the exposure of both infected individuals and medical professionals. Here we employed loop-mediated isothermal amplification and boiled swab samples (Direct Boil-LAMP method) from CL model mice to develop a simple and rapid diagnostic method for CL. The detection limit of this procedure was 1.0×10(3)parasites/mL. Accordingly, this substitutive diagnostic method should prove useful for mass screening. In addition, we discuss the potential advantages of using it, particularly in endemic regions where medical resources are limited.

Successful diagnosis of tuberculous lymphadenitis by loop-mediated isothermal amplification of cutaneous samples from an ulcerated surface lesion: a case report.

INTRODUCTION: Tuberculous lymphadenitis is the most frequent form of extrapulmonary tuberculous. Although nucleic acid amplification assays such as polymerase chain reaction have recently become mainstream techniques for diagnosing tuberculous lymphadenitis, they are still not routinely performed in developing countries because of their high costs and complicated procedures. CASE PRESENTATION: We describe a case of tuberculous lymphadenitis in a 79-year-old Japanese man who had been on continuous hemodialysis for end-stage renal disease. We employed loop-mediated isothermal amplification and the procedure for ultrarapid extraction to develop a fast and easy-to-perform procedure for diagnosing tuberculous lymphadenitis. CONCLUSIONS: The commercially available loop-mediated isothermal amplification assay kit and a rapid purification procedure enabled us to identify and amplify a Mycobacterium tuberculosis-specific gene within just 1.5 hours.