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Typhi and performed qPCR as described by Nga et al. As expected, we observed an inverse relationship between CFU and cycle threshold C t values. Typhi was detected by qPCR. Previous attempts to detect Salmonella in whole blood by MAMEF were unsuccessful due to congealing of erythrocytes and clotting factors under microwave heating results not shown.
The role of clotting factors in congealing was supported by the fact that when plasma and serum were microwave lysed, samples also congealed under exposure to microwave heat results not shown. We took advantage of the fact that approx. We tested microwave lysis of white blood cells spiked with Salmonella and suspended in various diluents. We also observed that when too little salt was present during microwave lysis, DNA release and fragmentation was suboptimal as evidenced by a DNA smear on an agarose gel with a peak band of approx.
Optimal conditions for microwave lysis were ultimately identified as white blood cells suspended in APF-LB previously used for microwave lysis of Salmonella as described by Tennant et al. Eventually, we were able to estimate the effectiveness of microwave lysing and DNA release by visually inspecting the cracking of the gold bow tie after microwave lysis with more cracking signalling maximal DNA release and fragmentation Fig.
Lysis was optimized by adjusting the microwave power and time until maximal DNA release and fragmentation to approx. We examined gold cracking and also verified fragment sizes using the Agilent Bioanalyzer Fig. As shown in Fig. As a result of complete removal of erythrocytes, we were able to avoid congealing under microwave heat and enable efficient lysis of samples.
Salt concentration-dependent fragmentation of DNA and gold cracking produced by microwave lysis. Microwave lysis of Salmonella. DNase and RNase treatment of Salmonella. However, when we attempted detection of a synthetic oriC oligonucleotide suspended in blood, MAMEF was not able to detect a fluorescent signal. When the spiked blood sample was diluted with an equal volume of PBS, a fluorescent signal was detected, presumably due to a reduction in the viscosity of blood.
After repeated experiments using diluted blood, we found that sometimes the blood would congeal during microwave heating, which rendered it impossible for the fluorescent signal to be detected.
We determined that it was necessary to remove the erythrocytes in order to avoid congealing under microwave radiation and to allow the fluorophore to be detected. Cognizant of the fact that the majority of Salmonella organisms in an infected individual reside in the mononuclear cells, we compared two fundamental approaches to obtain the mononuclear cells.
In the first approach we treated whole blood by various methods to lyse the erythrocytes and thereupon pelleted the white blood cells. In the alternative approach, we kept the erythrocytes intact, while separating out the mononuclear cell-rich buffy coat layer.
Typhi are found within mononuclear cells in a typhoid fever patient, when blood from a healthy human is spiked with Salmonella in vitro , only approx.
To overcome this experimental limitation, we spiked the white blood cells after treating the blood with various techniques. This provided a means to more accurately compare the efficiency of our various lysis and detection methods. It is attractive to have the ability to obtain both extracellular and intracellular Salmonella as is possible by treating blood with ammonium chloride, erythrocyte lysis buffer or water.
Moreover, these are inexpensive reagents and the methods are practical and fast. However, they suffer one possible limitation for developing country laboratories, namely, the separation of white blood cells and bacteria requires the use of a bench top centrifuge.
Even then, while all the intracellular organisms may be recovered, not all the extracellular organisms pellet at the low speed needed for optimal white blood cell separation.
Although water is a simple and inexpensive method for lysis, it is less well established and may cause premature lysis of white blood cells and their bacteria; release of DNA earlier than desired may result in loss of DNA when the supernatant is discarded. Overall, erythrocyte lysis buffer was our preferred method of treating whole blood. For the buffy coat methods, blood must be processed within a h period for optimal separation of the buffy coat. For the lymphocyte separation medium, in addition to requiring a bench top centrifuge, more skilled handling of small volumes of blood is necessary to prevent the disruption of the interface between the buffy coat layer and the erythrocytes.
Among the buffy coat extraction methods, dextran is the most promising because it is easy to perform, inexpensive, easy to handle and requires only an inexpensive, small microcentrifuge. However, it is highly effective only when used with fresh blood.
For utilization in field situations where blood may be collected from a remote site and transported for some hours to a blood processing laboratory, erythrocyte lysis buffer is the preferred method for isolation of white blood cells, as it does not require fresh blood for optimal separation. Despite the limitations inherent in each separation method, one major advantage common to all is that any volume of blood may be processed and the white blood cells resuspended in any volume necessary for detection by molecular methods such as MAMEF or PCR or quantitative real-time PCR.
This is particularly important in Salmonella infection in young and malnourished subjects from whom only small volumes of blood are typically collected to make a laboratory diagnosis. It is important to stress that testing white blood cells alone from typhoid fever patients does not decrease sensitivity, as has already been demonstrated by Wain et al. Typhi mixed with white blood cells from 2 ml of blood. We have also shown that we can effectively lyse Salmonella suspended in a buffy coat fraction in preparation for detection by MAMEF.
We conclude that adaptation of the erythrocyte lysis procedure has enabled improved sensitivity of detection by PCR and real-time PCR following DNA extraction and has allowed lysis and fragmentation of Salmonella using microwave radiation for future detection by MAMEF.
We are currently evaluating whether erythrocyte lysis and subsequent detection by real-time PCR is faster and more sensitive than blood culture in a typhoid endemic setting.
Importantly, these methods are economical and can be adapted for use in field studies using molecular methods. They may also be used for diagnosis in hospitals that lack blood culture machines and antisera for identification of Salmonella, but which are capable of performing molecular biology procedures.
The funder had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. Geddes possesses a patent for the microwave lysing technology. National Center for Biotechnology Information , U. Journal of Applied Microbiology. Published online Mar Author information Article notes Copyright and License information Disclaimer. Center for Biologics Evaluation and Research, U. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
This article has been cited by other articles in PMC. Abstract Aims Isolation of Salmonella Typhi from blood culture is the standard diagnostic for confirming typhoid fever but it is unavailable in many developing countries. Significance and Impact of the Study Adaptation of the blood lysis method represents a fundamental breakthrough that improves the sensitivity of DNA-based detection of Salmonella in blood.
Introduction Salmonella enterica serovars Typhi and Paratyphi A and B cause enteric fever, febrile illnesses that cannot be clinically distinguished from one another and which are difficult to differentiate from other causes of febrile illness Crump Water Two millilitres of whole blood were mixed with 18 ml of distilled water. Extraction of DNA from spiked white blood cells Two millilitres of whole blood was lysed using erythrocyte lysis buffer as described above.
Lysis of Salmonella by microwave radiation In order to focus microwaves, gold bowtie lysis triangles were deposited on glass slides as described previously Tennant et al. Results Salmonella uptake by white blood cells In patients with typhoid fever the majority of S. Open in a separate window.
Blood lysis We tested six techniques for separating white blood cells from whole blood. Table 1 Comparison of blood separation methods. Gel-based PCR detection of Salmonella To determine whether sensitivity of detection of Salmonella in blood could be improved by targeting white blood cells instead of using whole blood, we used conventional gel-based PCR.
Microwave lysis of Salmonella and fragmentation of DNA Previous attempts to detect Salmonella in whole blood by MAMEF were unsuccessful due to congealing of erythrocytes and clotting factors under microwave heating results not shown.
Table 3 Salt concentration-dependent fragmentation of DNA and gold cracking produced by microwave lysis. Conflict of interest Professor Christopher D. Separation of white blood cells. Typhoid fever and paratyphoid fever: Typhoid fever and the challenge of nonmalaria febrile illness in sub-saharan Africa. Global trends in typhoid and paratyphoid Fever. Estimating the incidence of typhoid fever and other febrile illnesses in developing countries. They are often found in small creeks, as well as billabongs and the edges of larger rivers.
They prefer water 1 to 2 m deep with aquatic weed and structure provided by rocks or sunken timber usually the latter. Like many other Murray-Darling native fish species, western carp gudgeon have crossed the Great Dividing Range through natural river capture events and are found in a number of East Coast drainages, from the Hunter River system in northern New South Wales to the Fitzroy River system in central Queensland.
Usually, their bodies are yellowish to clear with subtle, dark grey blotches, with semitranslucent fins. Males develop spectacular spawning colours in summer, namely a more metallic-bronze body colour with a red stripe through the caudal tail , anal, and spiny and soft dorsal fins. The red stripe through the anal and spiny and soft dorsal fins are topped with a tiny iridescent blue-white stripe, which are somewhat subtle on most of the fins, but are prominent and eye-catching on the spiny dorsal fin.
Western carp gudgeons spawn in summer, attaching eggs to aquatic weed in the shallows. They may have suffered from small variations in river level caused by river regulation, which exposes and destroys eggs laid in shallows.
They may be a critical food item for juvenile Murray cod , and likely are an important forage fish for larger fish species in many waterways. Western carp gudgeons have taken to lower-altitude, man-made lakes and impoundments in the Murray-Darling system well, and are very common in some. Western carp gudgeon make superb aquarium fish: They eagerly take live or frozen brine shrimp , very small invertebrates , and commercial micropellet fish feeds.
Curiously, they are unavailable in aquarium stores and remain the province of keen naturalists who capture their own. The specific name honours the German physician and zoologist Carl Benjamin Klunzinger , who, in , mistook this species for Eleotris cyprinoides. Western carp gudgeon are purportedly the most common of the carp gudgeons.
However, recent genetic research suggests that the carp gudgeons are a cryptic species complex composed of at least four species and many hybrids; their taxonomy is extremely complicated, unresolved, and not accurately reflected by current scientific and common names. Many researchers have now resorted to referring to them as simply "carp gudgeon Hypseleotris spp. Despite their common name, carp gudgeon are in no way related to carp Cyprinus carpio or other members of the Cyprinidae family.