Budget impact of next-generation sequencing for diagnosis of TB drug resistance in Moldova.

BACKGROUND Diagnosing drug resistance is critical for choosing effective TB treatment regimens. Next-generation sequencing (NGS) represents an alternative approach to conventional phenotypic drug susceptibility testing (pDST) for diagnosing TB drug resistance.METHODS We undertook a budget impact analysis estimating the costs of introduction and routine use of NGS in the Moldovan National TB Programme. We conducted an empirical costing study and collated price and operating characteristics for NGS platforms. We examined multiple NGS scenarios in comparison to the current approach (pDST) for pre-treatment drug resistance testing over 2021-2025.RESULTS Annual testing volume ranged from 912 to 1,926 patients. For the pDST scenario, we estimated total costs of US$362,000 (2021 USD) over the 5-year study period. Total costs for NGS scenarios ranged from US$475,000 to US$1,486,000. Lowest cost NGS options involved targeted sequencing as a replacement for pDST, and excluded individuals diagnosed as RIF-susceptible on Xpert® MTB/RIF. For all NGS scenarios, the majority (55-80%) of costs were devoted to reagent kits. Start-up costs of NGS were small relative to routine costs borne each year.CONCLUSION NGS adoption will require expanded resources compared to conventional pDST. Further work is required to better understand the feasibility of NGS in settings such as Moldova.

Direct detection of Coccidioides from Arizona soils using CocciENV, a highly sensitive and specific real-time PCR assay.

Coccidioides immitis and Coccidioides posadasii are soil fungi endemic to desert regions of the southwestern United States, and the causative agents of valley fever, or coccidioidomycosis. Studies have shown that the distribution of Coccidioides in soils is sporadic and cannot be explained by soil characteristics alone, suggesting that biotic and other abiotic factors should be examined. However, tools to reliably and robustly screen the large number of soils needed to investigate these potential associations have not been available. Thus, we developed a real-time polymerase chain reaction (PCR) assay for testing environmental samples by modifying CocciDx, an assay validated for testing clinical specimens to facilitate coccidioidomycosis diagnosis. For this study, we collected soil samples from previously established locations of C. posadasii in Arizona and new locations in fall 2013 and spring 2014, and screened the extracted DNA with the new assay known as CocciEnv. To verify the presence of Coccidioides in soil using an alternate method, we employed next generation amplicon sequencing targeting the ITS2 region. Results show our modified assay, CocciEnv, is a rapid and robust method for detecting Coccidioides DNA in complex environmental samples. The ability to test a large number of soils for the presence of Coccidioides is a much-needed tool in the understanding of the ecology of the organism and epidemiology of the disease and will greatly improve our understanding of this human pathogen.

Transmission of methicillin-resistant Staphylococcus aureus infection through solid organ transplantation: confirmation via whole genome sequencing.

We describe two cases of donor-derived methicillin-resistant Staphylococcus aureus (MRSA) bacteremia that developed after transplantation of organs from a common donor who died from acute MRSA endocarditis. Both recipients developed recurrent MRSA infection despite appropriate antibiotic therapy, and required prolonged hospitalization and hospital readmission. Comparison of S. aureus whole genome sequence of DNA extracted from fixed donor tissue and recipients' isolates confirmed donor-derived transmission. Current guidelines emphasize the risk posed by donors with bacteremia from multidrug-resistant organisms. This investigation suggests that, particularly in the setting of donor endocarditis, even a standard course of prophylactic antibiotics may not be sufficient to prevent donor-derived infection.

Quantitative competitive PCR as a technique for exploring flea-Yersina pestis dynamics.

We used a quantitative competitive polymerase chain reaction assay to quantify Yersinia pestis loads in fleas and bacteremia levels in mice that were used as sources of infectious blood meals for feeding the fleas. Xenopsylla cheopis, the Oriental rat flea, achieved higher infection rates, developed greater bacterial loads, and became infectious more rapidly than Oropsylla montana, a ground squirrel flea. Both flea species required about 10(6) Y. pestis cells per flea to be able to transmit to mice. Most fleas that achieved these levels, however, were incapable of transmitting. Our results suggest that at the time of flea feeding, host blood must contain > or = 10(6) bacteria/ml to result in detectable Y. pestis infections in these fleas, and > or = 10(7) bacteria/mL to cause infection levels sufficient for both species to eventually become capable of transmitting Y. pestis to uninfected mice. Yersinia pestis colonies primarily developed in the midguts of O. montana, whereas infections in X. cheopis often developed simultaneously in the proventriculus and the midgut. These findings were visually confirmed by infecting fleas with a strain of Y. pestis that had been transformed with the green fluorescent protein gene.

Quantities of Yersinia pestis in fleas (Siphonaptera: Pulicidae, Ceratophyllidae, and Hystrichopsyllidae) collected from areas of known or suspected plague activity.

We used a quantitative competitive polymerase chain reaction (PCR) (QC-PCR) to determine bacterial loads in 669 fleas collected in areas of confirmed and suspected plague epizootics. Fleas were collected out of rodent burrows (67.9%) and off of captured animals (24.1%) and rodent carcasses (8.1%). An initial PCR screening assay indicated that 12.1% (81/669) of all fleas were positive for Yersinia pestis. Fleas collected from burrows had significantly lower (chi2 = 264.9, P < 0.0001) infection rates (6.8%) but significantly higher (Student t-test, P < 0.0001) bacterial loads (mean = 10(5.6) Y. pestis per flea) than fleas collected off of rodent carcasses (infection rate = 92.6%; mean bacterial load = 10(4.8) Y. pestis per flea). None of the fleas collected off of captured animals were positive for Y. pestis by PCR, although seven of the 176 captured animals were serologically positive for Y. pestis.

PCR detection of Yersinia pestis in fleas: comparison with mouse inoculation.

The "gold standard" for identifying Yersinia pestis-infected fleas has been inoculation of mice with pooled flea material. Inoculated mice are monitored for 21 days, and those that die are further analyzed for Y. pestis infection by fluorescent-antibody assay and/or culture. PCR may provide a more rapid and sensitive alternative for identifying Y. pestis in fleas. To compare these assays, samples were prepared from 381 field-collected fleas. Each flea was analyzed individually by both PCR and mouse inoculation. Sixty of the 381 flea samples were positive for Y. pestis by PCR; 48 of these PCR-positive samples caused death in mice (80.0% agreement). None of the 321 PCR-negative samples caused death. Among the 12 mice that survived inoculation with PCR-positive samples, 10 were later demonstrated by serology or culture to have been infected with Y. pestis. This suggests that death of inoculated mice is less reliable than PCR as an indicator of the presence of Y. pestis in flea samples. Mouse inoculation assays produce results that are comparable to PCR only when surviving as well as dead mice are analyzed for infection. The rapidity and sensitivity (10 to 100 CFU of Y. pestis) of PCR suggest that it could serve as a useful alternative to mouse inoculation for routine plague surveillance and outbreak investigations.

Climatic and environmental patterns associated with hantavirus pulmonary syndrome, Four Corners region, United States.

To investigate climatic, spatial, temporal, and environmental patterns associated with hantavirus pulmonary syndrome (HPS) cases in the Four Corners region, we collected exposure site data for HPS cases that occurred in 1993 to 1995. Cases clustered seasonally and temporally by biome type and geographic location, and exposure sites were most often found in pinyon-juniper woodlands, grasslands, and Great Basin desert scrub lands, at elevations of 1,800 m to 2,500 m. Environmental factors (e.g., the dramatic increase in precipitation associated with the 1992 to 1993 El Niño) may indirectly increase the risk for Sin Nombre virus exposure and therefore may be of value in designing disease prevention campaigns.

Short report: Decrease in seroprevalence of antibodies to hantavirus in rodents from 1993-1994 hantavirus pulmonary syndrome case sites.

Rodent trapping was conducted at seven hantavirus pulmonary syndrome (HPS) case sites from June 1993 to March 1994 during the HPS outbreak in the southwestern United States. To determine if there were changes in the rodent population or the hantavirus seroprevalence in rodents since the HPS outbreak, rodents were trapped at the same sites three years later using the same trapping protocol. The trap success decreased from the numbers trapped during the outbreak, however, the number of Peromyscus, as a percentage of the total rodents captured, did not noticeably decrease. In addition, the seroprevalence of hantavirus antibodies in Peromyscus decreased significantly (P < 0.0001).

Patterns of association with host and habitat: antibody reactive with Sin Nombre virus in small mammals in the major biotic communities of the southwestern United States.

The distribution and prevalence of antibody reactive with Sin Nombre virus were determined in mammals in biotic communities of the southwestern United States. Small mammals (n = 3,069) of 69 species were trapped in nine communities from lower Sonoran desert to alpine tundra. Antibody was found in rodents from all communities (overall prevalence = 6.3%); prevalence was lowest at the altitudinal and climatic extremes (0.4% in desert and 2.0% in alpine tundra). Antibody occurred in 11% of 928 deer mice, 20% of 355 brush mice, 23% of 35 western harvest mice, and 12% of 24 Mexican voles. No infected deer mice were found in desert habitat; prevalence varied from 4% in chaparral to 17% in pinyon-juniper. Brush mice were frequently infected in chaparral and montane forest (25%). Seropositivity was higher in males and in heavier animals, suggesting horizontal transmission among adult males. Decreasing prevalence with age among the youngest deer mice suggests that infected dams confer passive immunity to pups.