Higher blood volumes improve the sensitivity of direct PCR diagnosis of blood stream tuberculosis among HIV-positive patients: an observation study.

BACKGROUND: Blood stream tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB) is common among HIV-positive patients, turning rapidly fatal unless detected and treated promptly. Blood culture is currently the standard test for the detection of MTB in whole blood but results take weeks; patients deteriorate markedly and often die before a diagnosis of blood stream TB is made. Rapid molecular tests on whole blood, with potential for same day diagnosis of blood stream TB usually show low sensitivity due to the problem of insufficient MTB DNA template when extraction is performed directly on low blood volumes. This study assessed the influence of blood volume on the sensitivity of a HyBeacon PCR assay-the FluoroType MTB (Hain Lifescience, Nehren, Germany) on direct detection of MTB in whole blood. METHODS: Prospective recruitment of HIV-positive patients with clinical suspicion of blood stream TB but not on anti-TB or HIV drug treatment was done. Venous blood samples were collected and DNA extracted using the MolYsis (Molzym, Bremen, Germany) methods; for study A, from duplicate 1 ml (42 patients) and for study B (31 patients) from 9 ml EDTA blood samples. The FluoroType MTB PCR assay targeting an IS6110 sequence was performed and results compared with blood culture. RESULTS: The diagnostic sensitivity and specificity of the FluoroType MTB PCR in study A was 33% and 97%, respectively. Corresponding values in study B were 71% and 96%, respectively. In both studies, one case each of blood culture-negative blood stream TB was detected with the FluoroType MTB PCR assay. The median time to positivity of blood culture was 20.1 (range 12-32) for study A and 19.9 days (range 15-30) for study B. CONCLUSION: Larger blood volumes (9 ml) improved and gave acceptable sensitivity of direct PCR diagnosis of blood stream TB.

Monitoring of patients supported by extracorporeal membrane oxygenation for systemic infections by broad-range rRNA gene PCR amplification and sequence analysis.

The rRNA gene PCR and sequencing test, SepsiTest, was compared with blood culture (BC) regarding the diagnosis of pathogens in 160 blood samples drawn from 28 patients during extracorporeal membrane oxygenation. With 45% of positive samples, SepsiTest was 13 to 75 h faster than BC. SepsiTest indicated bacteremias in 25% of patients who were BC negative.

Potential impact of cell-free DNA blood testing in the diagnosis of sepsis.

BACKGROUND: Classical blood culture testing is still the gold standard in correct and timely diagnosis of the responsible microorganisms in sepsis. CASE SUMMARY: In this case (a patient with a colon perforation and severe peritonitis with septic shock), an alternative approach (cell-free DNA next-generation sequencing from full blood samples, NGS) showed the responsible microorganisms, whereas the classical blood culture testing remainedstayed sterile. Interestingly, samples from the abdominal fluid showed the same bacteria as NGS. CONCLUSION: These findings may be interpreted as that the threshold for positive testing is lower through the molecular approach than through culture techniques; however, more studies are necessary to prove this theory.

Evaluation of commercial universal rRNA gene PCR plus sequencing tests for identification of bacteria and fungi associated with infectious endocarditis.

Two new commercially available universal rRNA gene PCR plus sequencing tests, SepsiTest and universal microbe detection (UMD; Molzym, Bremen, Germany), were evaluated using blood specimens and heart valves from 30 patients with suspected infectious endocarditis (IE). The sensitivity of PCR (85%) was nearly twice as high as that of culture (45%), which in 10/20 IE cases presumably stayed negative as a consequence of growth inhibition of the pathogens by antibiotics. Further, PCR provided the basis for reclassification of 5/10 non-IE cases into IE cases. Culture-negative infections were identified by PCR, including single infections due to streptococci and Gram-negative bacteria (Escherichia coli, Haemophilus parainfluenzae) and mixed infections involving two Gram-positive bacteria or Candida spp. with Gram-positive bacteria. The new commercial tests proved to be of value for the rapid diagnosis of IE, particularly in cases of culture-negative infections. Issues regarding the feasibility of these tests for routine use are discussed.

Detection of "Candidatus Neoehrlichia mikurensis" in two patients with severe febrile illnesses: evidence for a European sequence variant.

Recently, a new genus of Anaplasmataceae termed "Candidatus Neoehrlichia" was discovered in ticks and rodents. Here, we report on two patients who suffered from febrile bacteremia due to "Candidatus Neoehrlichia mikurensis" associated with thrombotic or hemorrhagic events. 16S rRNA and groEL gene sequencing provided evidence of three groups of sequence variants.

Diagnosis of bacteremia in whole-blood samples by use of a commercial universal 16S rRNA gene-based PCR and sequence analysis.

In a prospective, multicenter study of 342 blood samples from 187 patients with systemic inflammatory response syndrome, sepsis, or neutropenic fever, a new commercial PCR test (SepsiTest; Molzym) was evaluated for rapid diagnosis of bacteremia. The test comprises a universal PCR from the 16S rRNA gene, with subsequent identification of bacteria from positive samples by sequence analysis of amplicons. Compared to blood culture (BC), the diagnostic sensitivity and specificity of the PCR were 87.0 and 85.8%, respectively. Considering the 34 BC-positive patients, 28 were also PCR positive in at least one of the samples, resulting in a patient-related sensitivity of 82.4%. The concordance of PCR and BC for both positive and negative samples was (47 + 247)/342, i.e., 86.0%. In total, 31 patients were PCR/sequencing positive and BC negative, in whom the PCR result was judged as possible or probable to true bacteremia in 25. In conclusion, the PCR approach facilitates the detection of bacteremia in blood samples within a few hours. Despite the indispensability of BC diagnostics, the rapid detection of bacteria by SepsiTest appears to be a valuable tool, allowing earlier pathogen-adapted antimicrobial therapy in critically ill patients.

Ipsilateral parenchymal hemorrhage after hemicraniectomy in a patient suffering from malignant middle cerebral artery infarction.

INTRODUCTION: Early hemicraniectomy reduces mortality in malignant middle cerebral artery (MCA) infarctions to 16%, although the benefit on functional outcome is still unclear. We treat patients with malignant MCA infarction younger than 60 years. Epidural or subgaleal hemorrhages are relatively common complications. Only 1 trial described parenchymal hemorrhage as a complication of hemicraniectomy. CASE REPORT: A 55-year-old man was admitted to our stroke unit with right-sided hemiparesis and aphasia. NIHSS was 18; GCS, 3. The initial CT showed hypoattenuation in the basal ganglia and the insular ribbon on the left side. CT-angiography showed M1 occlusion. Despite thrombolysis with rtPA in the 3-hour time window, no recanalization was achieved. We performed early hemicraniectomy. Additionally, we started mild hypothermia and deep sedation to prevent increasing cerebral edema. Control CT on day 2 showed intracerebral hemorrhage and an obstructive hydrocephalus because of intraventricular blood. To avoid herniation, the patient received an extraventricular drainage (EVD). Thereafter, the intracerebral pressure remained stable. The patient was discharged, with a NIHSS of 18 and GCS of 10. CONCLUSION: The reported patient is the first in the literature that suffered from deep hemorrhage after hemicraniectomy. Hemorrhagic transformation might be a risk factor for clinically relevant hemorrhage after hemicraniectomy.

Bacterial and fungal DNA extraction from blood samples: automated protocols.

Automation in DNA isolation is a necessity for routine practice employing molecular diagnosis of infectious agents. To this end, the development of automated systems for the molecular diagnosis of microorganisms directly in blood samples is at its beginning. Important characteristics of systems demanded for routine use include high recovery of microbial DNA, DNA-free containment for the reduction of DNA contamination from exogenous sources, DNA-free reagents and consumables, ideally a walkaway system, and economical pricing of the equipment and consumables. Such full automation of DNA extraction evaluated and in use for sepsis diagnostics is yet not available. Here, we present protocols for the semiautomated isolation of microbial DNA from blood culture and low- and high-volume blood samples. The protocols include a manual pretreatment step followed by automated extraction and purification of microbial DNA.

Bacterial and fungal DNA extraction from blood samples: manual protocols.

A critical point of molecular diagnosis of systemic infections is the method employed for the extraction of microbial DNA from blood. A DNA isolation method has to be able to fulfill several fundamental requirements for optimal performance of diagnostic assays. First of all, low- and high-molecular-weight substances of the blood inhibitory to downstream analytical reactions like PCR amplification have to be removed. This includes human DNA which is a known source of false-positive results and factor decreasing the analytical sensitivity of PCR assays by unspecific primer binding. At the same time, even extremely low amounts of microbial DNA need to be supplied to molecular diagnostic assays in order to detect low pathogen loads in the blood. Further, considering the variety of microbial etiologies of sepsis, a method should be capable of lysing Gram-positive, Gram-negative, and fungal organisms. Last, extraction buffers, reagents, and consumables have to be free of microbial DNA which leads to false-positive results. Here, we describe manual methods which allow the extraction of microbial DNA from small- and large-volume blood samples for the direct molecular analysis of pathogen.

Activity and DNA contamination of commercial polymerase chain reaction reagents for the universal 16S rDNA real-time polymerase chain reaction detection of bacterial pathogens in blood.

Universal 16S rRNA gene polymerase chain reaction (PCR) is a promising means of detecting bacteremia. Among other factors, the PCR reagents play a prominent role for obtaining a high sensitivity of detection. The reagents are ideally optimized with respect to the amplifying activity and absence of contaminating DNA. In this study, it was shown in a universal 16S rDNA real-time PCR assay that commercial PCR reagents can vary greatly among each other in these characters. Only 1 of the 5 reagents tested met the criteria of sensitive detection of pathogen DNA with a minimum of false-positive results. The reagent was validated by the detection of pathogens at low titers using bacterial DNA extracted from blood that was spiked with various Gram-positive and Gram-negative bacteria.