P. Tattevin

Simultaneous determination of 12 beta-lactam antibiotics in human plasma by high-performance liquid chromatography with UV detection: application to therapeutic drug monitoring.

ABSTRACT A rapid and specific high-performance liquid chromatography method with UV detection (HPLC-UV) for the simultaneous determination of 12 beta-lactam antibiotics (amoxicillin, cefepime, cefotaxime, ceftazidime, ceftriaxone, cloxacillin, imipenem, meropenem, oxacillin, penicillin G, piperacillin, and ticarcillin) in small samples of human plasma is described. Extraction consisted of protein precipitation by acetonitrile. An Atlantis T3 analytical column with a linear gradient of acetonitrile and a pH 2 phosphoric acid solution was used for separation. Wavelength photodiode array detection was set either at 210 nm, 230 nm, or 298 nm according to the compound. This method is accurate and reproducible (coefficient of variation [CV] < 8%), allowing quantification of beta-lactam plasma levels from 5 to 250 μg/ml without interference with other common drugs. This technique is easy to use in routine therapeutic drug monitoring of beta-lactam antibiotics.

A small RNA controls a protein regulator involved in antibiotic resistance in Staphylococcus aureus

The emergence of Staphylococcus aureus strains that are resistant to glycopeptides has led to alarming scenarios where serious staphylococcal infections cannot be treated. The bacterium expresses many small regulatory RNAs (sRNAs) that have unknown biological functions for the most part. Here we show that an S. aureus sRNA, SprX (alias RsaOR), shapes bacterial resistance to glycopeptides, the invaluable treatments for Methicillin-resistant staphylococcal infections. Modifying SprX expression levels influences Vancomycin and Teicoplanin glycopeptide resistance. Comparative proteomic studies have identified that SprX specifically downregulates stage V sporulation protein G, SpoVG. SpoVG is produced from the yabJ-spoVG operon and contributes to S. aureus glycopeptide resistance. SprX negatively regulates SpoVG expression by direct antisense pairings at the internal translation initiation signals of the second operon gene, without modifying bicistronic mRNA expression levels or affecting YabJ translation. The SprX and yabJ-spoVG mRNA domains involved in the interaction have been identified, highlighting the importance of a CU-rich loop of SprX in the control of SpoVG expression. We have shown that SpoVG might not be the unique SprX target involved in the glycopeptide resistance and demonstrated that the regulation of glycopeptide sensitivity involves the CU-rich domain of SprX. Here we report the case of a sRNA influencing antibiotic resistance of a major human pathogen.

Respiratory Manifestations of Leptospirosis: A Retrospective Study

We retrospectively reviewed 34 consecutive patients with serologically confirmed leptospirosis admitted during years 1992–2002. Nine patients (26.5%) had respiratory symptoms on admission including cough (n = 4), shortness of breath (n = 4), cyanosis (n = 2), and hemoptysis (n = 1). Six patients had pulmonary radiographic findings including (1) diffuse, ill-defined, ground-glass density (n = 3); (2) diffuse alveolar opacities (n = 2); and (3) small nodular density (n = 1). Male/female ratio was 8/1 and mean age was 47 years. Seven patients reported their exposure source including hunting (n = 2), fishing (n = 2), fresh water swimming (n = 2), and canoeing (n = 1). All patients had fever (mean = 40.1°C). Other common symptoms were headache (n = 4), vomiting (n = 3), and myalgia (n = 3). Biological abnormalities included elevated liver enzymes (n = 8), proteinuria (n = 7), lymphopenia (n = 6), hematuria (n = 5), renal failure (n = 4), anemia (n = 4), and elevated neutrophil count (n = 4). PaO2 was measured for 3 patients while they were breathing room air (32, 55, and 66xa0mmHg). Suspected diagnosis on admission included leptospirosis (n = 2), bacterial pneumonia (n = 2), intoxication, influenza, viral hepatitis, biliary tract lithiasis, and rapidly progressive glomerulonephritis (one patient each). The first serologic testing for leptospirosis was positive for 5 patients (55%). Serovar was presumptively identified for 7 patients: Australis (n = 3), Grippotyphosa (n = 2), and Icterohaemorrhagiae (n = 2). Seven patients were treated with penicillin; two patients received no antibiotics. All patients were cured. In conclusion, patients with leptospirosis may present predominantly with nonspecific pulmonary symptoms. In these patients, leptospirosis must be suspected when there is a potential exposure to rats, especially in case of high-grade fever, myalgia, hepatitis, and renal abnormalities.

Human herpes virus co-infection is associated with mortality in HIV-negative patients with Pneumocystis jirovecii pneumonia.

The purpose of this investigation was to characterize the management and prognosis of severe Pneumocystis jirovecii pneumonia (PJP) in human immunodeficiency virus (HIV)-negative patients. An observational cohort study of HIV-negative adults with PJP documented by bronchoalveolar lavage (BAL) through Gomori–Grocott staining or immunofluorescence, admitted to one intensive care unit (ICU) for acute respiratory failure, was undertaken. From 1990 to 2010, 70 patients (24 females, 46 males) were included, with a mean age of 58.6u2009±u200918.3xa0years. The mean Simplified Acute Physiology Score (SAPS)-II was 36.9u2009±u200920.4. Underlying conditions included hematologic malignancies (nu2009=u200921), vasculitis (nu2009=u200913), and solid tumors (nu2009=u200913). Most patients were receiving systemic corticosteroids (nu2009=u200963) and cytotoxic drugs (nu2009=u200951). Not a single patient received trimethoprim–sulfamethoxazole as PJP prophylaxis. Endotracheal intubation (ETI) was required in 42 patients (60.0xa0%), including 38 with acute respiratory distress syndrome (ARDS). In-ICU mortality was 52.9xa0% overall, reaching 80.9xa0% and 86.8xa0%, respectively, for patients who required ETI and for patients with ARDS. In the univariate analysis, in-ICU mortality was associated with SAPS-II (pu2009=u20090.0131), ARDS (pu2009<u20090.0001), shock (pu2009<u20090.0001), and herpes simplex virus (HSV) or cytomegalovirus (CMV) on BAL (pu2009=u20090.0031). In the multivariate analysis, only ARDS was associated with in-ICU mortality (odds ratio [OR] 23.4 [4.5–121.9], pu2009<u20090.0001). PJP in non-HIV patients remains a serious disease with high in-hospital mortality. Pulmonary co-infection with HSV or CMV may contribute to fatal outcome.

A retrospective study of 230 consecutive patients hospitalized for presumed travel-related illness (2000–2006)

A good knowledge of morbidity profiles among ill-returned travelers is necessary in order to guide their management. We reviewed the medical charts of 230 patients hospitalized in one infectious diseases department in France for presumed travel-related illnesses. The male-to-female ratio was 1.6 and the median age was 33xa0years (interquartile range [IQR], 25–50). Most patients (70.9%) were returning from sub-Saharan Africa. The median duration of travel was 28xa0days (IQR, 15–60) and the median time from return of travel to hospitalization was 13xa0days (IQR, 7–21). Malaria was the most frequent diagnosis (49.1%), which was especially encountered in patients returning from sub-Saharan Africa (95.6%), without adequate chemoprophylaxis (78.2%). Imported diseases at risk of secondary transmission were also diagnosed, including pulmonary tuberculosis (nu2009=u20098), viral hepatitis (nu2009=u20098), typhoid fever (nu2009=u20096), human immunodeficiency virus (HIV) (six new diagnosis), non-typhoid salmonellosis (nu2009=u20095), severe acute respiratory syndrome, and Crimean-Congo hemorrhagic fever. This study underlines the need to maintain tropical expertise for infectious diseases physicians, even in Europe.

Distinctive features between community-acquired pneumonia (CAP) due to Chlamydophila psittaci and CAP due to Legionella pneumophila admitted to the intensive care unit (ICU)

The spectrum of community-acquired pneumonia (CAP) due to Chlamydophila psittaci ranges from mild, self-limited CAP, to acute respiratory failure. We performed a retrospective study of 13 consecutive patients with CAP due to C. psittaci and 51 patients with legionellosis admitted in one intensive care unit (ICU) (1993–2011). As compared to patients with legionellosis, patients with psittacosis were younger (median age 48 [38–59] vs. 60 [50–71] years, pu2009=u20090.007), less frequently smokers (38xa0vs. 79xa0%, pu2009<u20090.001), with less chronic disease (15 vs. 57xa0%, pu2009=u20090.02), and longer duration of symptoms before admission (median 6 [5–13] vs. 5 [3–7] days, pu2009=u20090.038). They presented with lower Simplified Acute Physiology Score II (median 28 [19–38] vs. 39 [28–46], pu2009=u20090.04) and less extensive infiltrates on chest X-rays (median 2 [1–3] vs. 3 [3–4] lobes, pu2009=u20090.007). Bird exposure was mentioned in 100xa0% of psittacosis cases, as compared to 5.9xa0% of legionellosis cases (pu2009<u20090.0001). Extrapulmonary manifestations, biological features, and mortality (15.4 vs. 21.6xa0%, pu2009=u20090.62) were similar in both groups. In conclusion, severe psittacosis shares many features with severe legionellosis, including extrapulmonary manifestations, biological features, and outcome. Psittacosis is an important differential diagnosis for legionellosis, especially in cases of bird exposure, younger age, and more limited disease progression over the initial few days.

Feedback on difficulties raised by the interpretation of serological tests for the diagnosis of Lyme disease

OBJECTIVESnWe had for objectives: i) to evaluate the accuracy of serologic testing for Lyme borreliosis performed in a private medical laboratory (PML); ii) to evaluate the impact of these tests on the practices of infectious diseases specialists (IDS).nnnPATIENTS AND METHODnThis study was performed in two steps: i) retrospective study of patients followed in a university hospital infectious diseases outpatient clinic for suspected Lyme borreliosis, tested (ELISA and Western blot) by both the PML and the National Reference Center (NRC); ii) national survey on IDS practices concerning patients consulting for suspected Lyme borreliosis.nnnRESULTSnBetween July 2008 and July 2011, 128 patients consulting for suspected Lyme borreliosis were tested by both laboratories. Serological tests came back positive in 91% of cases from the PML versus 8% of cases from the NRC. Lyme borreliosis was the IDSs final diagnosis for 3.6% of patients. The survey on practices revealed that: i) the modal duration of consultation for suspected Lyme borreliosis was 30-60 minutes; ii) for 33% of patients, serologic test results performed at the PML were the only reason to suspect Lyme borreliosis; iii) 60% of patients had no indication for antibiotics.nnnCONCLUSIONnThe serological test performed in the PML were positive most of the time, but were not confirmed by tests performed at the NRC. This discrepancy lead to multiple and prolonged consultations in infectious diseases clinics, and discordance in the indications for antibiotics.

Pneumocystose chez les patients immunodéprimés non infectés par le VIH

Pneumocystis jiroveci (formerly P.xa0carinii) is an opportunistic fungus responsible for pneumonia in immunocompromised patients. Pneumocystosis in non-HIV-infected patients differs from AIDS-associated pneumocystosis in mostly two aspects: diagnosis is more difficult, and prognosis is worse. Hence, efforts should be made to target immunocompromised patients at higher risk of pneumocystosis, so that they are prescribed long-term, low-dose, trimethoprime-sulfamethoxazole, highly effective for pneumocystosis prophylaxis. Patients at highest risk include those with medium and small vessels vasculitis, lymphoproliferative B disorders (chronic or acute lymphocytic leukaemia, non-Hodgkin lymphoma), and solid cancer on long-term corticosteroids. Conversely, widespread use of prophylaxis in all patients carrier of inflammatory diseases on long-term corticosteroids is not warranted. The management of pneumocystosis in non-AIDS immunocompromised patients follows the rules established for AIDS patients. The diagnosis relies on the detection of P.xa0jiroveci cyst on respiratory samples, while PCR does not reliably discriminate infection from colonization, in 2015. High-doses trimethoprim-sulfamethoxazole is, by far, the treatment of choice. The benefit of adjuvant corticosteroid therapy for hypoxic patients, well documented in AIDS patients, has a much lower level of evidence in non-HIV-infected patients, most of them being already on corticosteroid by the time of pneumocystosis diagnosis anyway. However, based on its striking impact on morbi-mortality in AIDS patients, adjuvant corticosteroid is recommended in hypoxic, non-HIV-infected patients with pneumocystosis by many experts and scientific societies.

An in vivo reporter assay for sRNA-directed gene control in Gram-positive bacteria: identifying a novel sRNA target in Staphylococcus aureus

Abstract Bacterial small regulatory RNAs (sRNAs) play a major role in the regulation of various cellular functions. Most sRNAs interact with mRNA targets via an antisense mechanism, modifying their translation and/or degradation. Despite considerable progresses in discovering sRNAs in Gram-positive bacteria, their functions, for the most part, are unknown. This is mainly due to difficulties in identifying their targets. To aid in the identification of sRNA targets in Gram-positive bacteria, we set up an in vivo method for fast analysis of sRNA-mediated post-transcriptional control at the 5΄ regions of target mRNAs. The technology is based on the co-expression of an sRNA and a 5΄ sequence of an mRNA target fused to a green fluorescent protein (GFP) reporter. The system was challenged on Staphylococcus aureus, an opportunistic Gram-positive pathogen. We analyzed several established sRNA–mRNA interactions, and in addition, we identified the ecb mRNA as a novel target for SprX2 sRNA. Using our in vivo system in combination with in vitro experiments, we demonstrated that SprX2 uses an antisense mechanism to prevent ecb mRNA translation initiation. Furthermore, we used our reporter assay to validate sRNA regulations in other Gram-positive organisms, Bacillus subtilis and Listeria monocytogenes. Overall, our method is broadly applicable to challenge the predicted sRNA–mRNA interactions in Gram-positive bacteria.

[Pneumocystosis in non-HIV-infected immunocompromised patients].

Pneumocystis jiroveci (formerly P.xa0carinii) is an opportunistic fungus responsible for pneumonia in immunocompromised patients. Pneumocystosis in non-HIV-infected patients differs from AIDS-associated pneumocystosis in mostly two aspects: diagnosis is more difficult, and prognosis is worse. Hence, efforts should be made to target immunocompromised patients at higher risk of pneumocystosis, so that they are prescribed long-term, low-dose, trimethoprime-sulfamethoxazole, highly effective for pneumocystosis prophylaxis. Patients at highest risk include those with medium and small vessels vasculitis, lymphoproliferative B disorders (chronic or acute lymphocytic leukaemia, non-Hodgkin lymphoma), and solid cancer on long-term corticosteroids. Conversely, widespread use of prophylaxis in all patients carrier of inflammatory diseases on long-term corticosteroids is not warranted. The management of pneumocystosis in non-AIDS immunocompromised patients follows the rules established for AIDS patients. The diagnosis relies on the detection of P.xa0jiroveci cyst on respiratory samples, while PCR does not reliably discriminate infection from colonization, in 2015. High-doses trimethoprim-sulfamethoxazole is, by far, the treatment of choice. The benefit of adjuvant corticosteroid therapy for hypoxic patients, well documented in AIDS patients, has a much lower level of evidence in non-HIV-infected patients, most of them being already on corticosteroid by the time of pneumocystosis diagnosis anyway. However, based on its striking impact on morbi-mortality in AIDS patients, adjuvant corticosteroid is recommended in hypoxic, non-HIV-infected patients with pneumocystosis by many experts and scientific societies.