Multicentre study highlighting clinical relevance of new high-throughput methodologies in molecular epidemiology of Pneumocystis jirovecii pneumonia.

Pneumocystis jirovecii causes severe interstitial pneumonia (PcP) in immunosuppressed patients. This multicentre study assessed the distribution frequencies of epidemiologically relevant genetic markers of P. jirovecii in different geographic populations from Portugal, the USA, Spain, Cuba and Mozambique, and the relationship between the molecular data and the geographical and clinical information, based on a multifactorial approach. The high-throughput typing strategy for P. jirovecii characterization consisted of DNA pooling using quantitative real-time PCR followed by multiplex-PCR/single base extension. The frequencies of relevant P. jirovecii single nucleotide polymorphisms (mt85, SOD110, SOD215, DHFR312, DHPS165 and DHPS171) encoded at four loci were estimated in ten DNA pooled samples representing a total of 182 individual samples. Putative multilocus genotypes of P. jirovecii were shown to be clustered due to geographic differences but were also dependent on clinical characteristics of the populations studied. The haplotype DHFR312T/SOD110C/SOD215T was associated with severe AIDS-related PcP and high P. jirovecii burdens. The frequencies of this genetic variant of P. jirovecii were significantly higher in patients with AIDS-related PcP from Portugal and the USA than in the colonized patients from Portugal, and Spain, and children infected with P. jirovecii from Cuba or Mozambique, highlighting the importance of this haplotype, apparently associated with the severity of the disease and specific clinical groups. Patients from the USA and Mozambique showed higher rates of DHPS mutants, which may suggest the circulation of P. jirovecii organisms potentially related with trimethoprim-sulfamethoxazole resistance in those geographical regions. This report assessed the worldwide distribution of P. jirovecii haplotypes and their epidemiological impact in distinct geographic and clinical populations.

Diagnosis of Pneumocystis pneumonia: evaluation of four serologic biomarkers.

The diagnosis of Pneumocystis pneumonia (PCP) relies on microscopic visualization of Pneumocystis jirovecii organisms or DNA detection in pulmonary specimens. This study aimed to assess the usefulness of (1-3)-ß-d-glucan (BG), Krebs von den Lungen-6 antigen (KL-6), lactate dehydrogenase (LDH) and S-adenosyl methionine (SAM) as serologic biomarkers in the diagnosis of PCP. Serum levels of BG, KL-6, LDH and SAM were investigated in 145 Portuguese patients, 50 patients from the Netherlands, 25 Spanish patients and 40 Portuguese blood donors. Data on clinical presentation, chest imaging and gasometry tests were available. PCP cases were confirmed by microscopy and PCR techniques. A cost-effectiveness analysis was performed. BG was found to be the most reliable serologic biomarker for PCP diagnosis, followed by KL-6, LDH and SAM. The BG/KL-6 combination test was the most accurate serologic approach for PCP diagnosis, with 94.3% sensitivity and 89.6% specificity. Although less sensitive/specific than the reference standard classic methods based on bronchoalveolar lavage followed by microscopic or molecular detection of P. jirovecii organisms, the BG/KL-6 test may provide a less onerous procedure for PCP diagnosis, as it uses a minimally invasive and inexpensive specimen (blood), which may be also a major benefit for the patient's care. The BG/KL-6 combination test should be interpreted within the clinical context, and it may be used as a preliminary screening test in patients with primary suspicion of PCP, or as an alternative diagnostic procedure in patients with respiratory failure or in children, avoiding the associated risk of complications by the use of bronchoscopy.

(1-3)-beta-D-glucan in association with lactate dehydrogenase as biomarkers of Pneumocystis pneumonia (PcP) in HIV-infected patients.

Pneumocystis pneumonia (PcP) is a major HIV-related illness caused by Pneumocystis jirovecii. Definitive diagnosis of PcP requires microscopic detection of P. jirovecii in pulmonary specimens. The objective of this study was to evaluate the usefulness of two serum markers in the diagnosis of PcP. Serum levels of (1-3)-beta-d-glucan (BG) and lactate dehydrogenase (LDH) were investigated in 100 HIV-positive adult patients and 50 healthy blood donors. PcP cases were confirmed using indirect immunofluorescence with monoclonal anti-Pneumocystis antibodies and nested-PCR to amplify the large subunit mitochondrial rRNA gene of P. jirovecii in pulmonary specimens. BG and LDH levels in serum were measured using quantitative microplate-based assays. BG and LDH positive sera were statistically associated with PcP cases (P ≤ 0.001). Sensitivity, specificity, positive/negative predictive values (PPV/NPV), and positive/negative likelihood ratios (PLR/NLR) were 91.3 %, 61.3 %, 85.1 %, 79.2 %, 2.359, and 0.142, respectively, for the BG kit assay, and 91.3 %, 35.5 %, 75.9 %, 64.7 %, 1.415 and 0.245, respectively, for the LDH test. Serologic markers levels combined with the clinical diagnostic criteria for PcP were evaluated for their usefulness in diagnosis of PcP. The most promising cutoff levels for diagnosis of PcP were determined to be 400 pg/ml of BG and 350 U/l of LDH, which combined with clinical data presented 92.8 % sensitivity, 83.9 % specificity, 92.8 % PPV, 83.9 % NPV, 5.764 PLR and 0.086 NLR (P < 0.001). This study confirmed that BG is a reliable indicator for detecting P. jirovecii infection. The combination between BG/LDH levels and clinical data is a promising alternative approach for PcP diagnosis.

Spontaneous rupture of the spleen as presenting event in infectious mononucleosis.

We report the case of a patient who underwent a life-saving splenectomy after spontaneous rupture of the spleen. The patient had no history of recent trauma and developed obvious clinical signs of infectious mononucleosis only 5 d after the splenectomy. Also of note was the late seroconversion in this patient.

Cellular immune responses to Mycobacterium tuberculosis in a patient with Takayasu's arteritis.

INTRODUCTION: Takayasu's arteritis (TA) is a disease of unknown aetiology, characterized histologically by an inflammatory cell infiltrate that affects all layers of the arterial wall. Its association with tuberculosis (TB) was described 50 years ago, based on the presence of Langhan's giant cells and granulomas similar to those found in tuberculous lesions. The presence of TB in patients with TA well as been reported in several studies as well as a positive tuberculous response, but these associations could be fortuitous in countries where TB is endemic. Recent studies have shown that patients with TA have a heightened humoral response to mycobacterial antigens including the 65 kDa fraction, a heat shock protein (HSP) that has also been found to be expressed in the arterial wall of patients with TA. The purpose of this study was to determine lymphoproliferative response and interferon-gamma (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) stimulated by live Mycobacterium tuberculosis (Mtb) H37Rv and a panel of mycobacterial antigens, in the hope of contributing to a better understanding of the cellular immune responses to Tuberculosis in Takayasu's arteritis. MATERIAL AND METHODS: Standard lymphoproliferation tests and IFN-gamma determination (ELISA) were performed in a 47-year old black man who fulfilled criteria for TA and 10 healthy controls, BCG vaccinated, Mantoux positive. The following were used: Mtb H37Rv, Purified Protein Derivative (PPD), purified 30 kDa, recombinant M. bovis BCG 10 kDa, 38 kDa, 65 kDa, 70 kDa, Short Term-Culture Filtrate Proteins (ST-CFP), Mid Term-Culture Filtrate Proteins (MT-CFP) obtained from H37Rv and phytohemaglutinin (PHA) as mitogen for positive control. RESULTS: PBMC from the patient with TA when compared to the mean values of the 10 healthy donors showed decreased proliferation in response to all antigens, with the exception of 65 kDa. The TA patient showed a similar pattern of IFN-gamma production to that obtained with control donors, with the exception of higher IFN-gamma production in response to ST-CFP and MT-CFP. CONCLUSIONS: We have shown reactivity of peripheral lymphocytes to HSP 65 kDa and a trend towards higher production of IFN-gamma in response to ST-CFP and MT-CFP in a patient with TA. These facts, together with the already established heightened humoral response, strengthens the association between TB and TA. However, HSP 65 kDa is not specific to TB and we conclude that similar studies using lymphocytes obtained from the arterial wall of TA patients may help to clarify the role of mycobacterial infection in Takayasu's arteritis.

[Cellular immune response to mycobacterial antigens]. / Respostas imuno-celulares a antigénios micobacterianos.

The authors studied the immunological response before and after beginning anti-tuberculous therapy, of a previously health, HIV negative, 22 year old black male, from the Republic of Cape Verde. The patient had multiple vertebral bony lesions associated to subcutaneous abscesses. As immunological markers of antigen recognition, we measured blastogenic and cytotoxic responses and gamma-IFN secretion towards 30 kD, 65 kD, filtrate proteins of M. tuberculosis, M. tuberculosis (H37Rv) and PPD cultures. To characterise the role of cytokines during infection, expression of mRNA for gamma-IFN, IL-4 and IL-10 was also analysed. A slight increase of lymphocyte proliferation and gamma-IFN production was seen in response to purified protein derivative (PPD) and short term culture filtrate proteins (ST-CFP), after one month of therapy. More significant, was the increase in M. tuberculosis and PPD-specific cytolytic T lymphocyte response after one one month of treatment. After 6 months of treatment, blastogenic and cytotoxic responses and gamma-IFN production were considerably higher toward the antigen panel. The CD4/CD8 ratio increased from 0.7 to 1.4 after treatment. We observed that ST-CFP and MT-CFP induced increasingly higher lymphoproliferation and gamma-IFN production, confirming their role in the protective immune responses to M. tuberculosis. The reduced immune responses in the peripheral blood of this patient probably reflect a high activity in the local sites of infection. This case of disseminated tuberculosis infection maybe related to nutritional or social factors or may represent an example of reduced in ate resistance against tuberculosis infection.

[The effect of technopathologically limited stress factors on the fertility of cattle]. / Einfluss technopathiebedingter Stressfaktoren auf die Fruchtbarkeit des Rindes.

The influence of purulent claw-diseases on the development of uterus infections, its involution and ovary dysfunctions (acyclia, cysts) was examined. It was shown that A. pyogenes, Ps. aeruginosa, Fus. necrophorum and other microorganisms involved in these lesions are potential sources for the infections of the uterus. In comparison with the controls the frequency of endometritis was twice in the group with claw-problems. Similar differences existed in the involution-time of the uterus or its atonia, acyclia and the development of ovarian cysts (p less than 0.01). The Index of insemination was elevated by about 65%.

Ondine curse and neurocristopathy.

Two newborns, 1 male and 1 female, had both Ondine curse, also known as congenital, central hypoventilation syndrome, and Hirschsprung disease. Both infants demonstrated insufficient respiration while asleep and normal respiration when awake. The lesser affected child had an otherwise normal neurologic examination, but suffered from seizures. He died at 18 months of age; neuropathologic examination of the brain was unremarkable. The girl had a severe and ultimately fatal form of this disorder and manifested a variety of neurologic abnormalities indicative of developmental failure of the neural crest-derived tissues. These abnormalities included unreactive pupils and deafness. She died at 40 days of age; autopsy permission was denied. The etiology of sleep apnea is not known. Mechanisms of central integration may be abnormal but the association with neural crest maldevelopment implicates the peripheral nervous system.

Response to resistive loading in the newborn piglet.

The diaphragmatic force generation and electromyographic response to long-term (1 h) inspiratory resistive loading was examined in the newborn piglet during the 3rd postnatal wk of life. Minute ventilation decreased to approximately 50% of baseline level within 5 min of imposition of a severe resistive load and remained at this level for the duration of loading. The decrease in ventilation was secondary to a fall in tidal volume at a constant frequency. There was a significant increase in central nervous system output to the diaphragm as manifested by integrated diaphragmatic electromyogram. Progressive augmentation of this index of central drive continued throughout the period of loading. Functional residual capacity fell significantly by 60 min of inspiratory resistive loading. This strategy should allow greater force generation by placing the diaphragm at a more optimal length-tension relationship. However, the force generating capability of the diaphragm was compromised as assessed by force-frequency curve analysis. These results suggest that the diaphragm of the neonatal piglet fatigues during prolonged inspiratory resistive loading.

The effect of gastric banding on body weight in experimental animals.

Recently a new method of gastroplasty has been used in operative treatment of extreme obesity in humans. It depends on putting on the stomach an inabsorbable band in order to form two reservoirs: one small upper, the other large lower, and a narrow passage connecting them, without the necessity to open the stomach. Before using this method on humans it has been experimentally examined on 19 pigs. Gastric banding has little effect on the decrease of body weight, it has some effect only in the first 3-6 weeks. Bands put on the stomach move inside it because of the excessive built-up of pressure in the upper reservoir. The reservoirs vanish. Animals quickly put up weight. The pathological mechanism of moving the band inside the stomach can be explained by the presence of two processes: one reperative, on the serose layer, the other destructive, into the stomach. These findings prove that in operative treatment of extreme obesity in humans it seems essential not to allow the pressure to build up excessively in the upper stomach reservoir.

Massive congenital intracranial teratoma: a report of two cases.

Although intracranial teratoma is a well-recognized entity in the differential diagnosis of pediatric brain tumors, massive congenital intracranial teratoma replacing the cerebral hemispheres of a neonate has seldom been reported. We describe two such instances that histologically exhibit predominantly neuroepithelial differentiation. In 1 case serial prenatal ultrasonography revealed ventricular dilatation prior to identification of the lesion. Theories of pathogenesis are briefly discussed.

Bronchopulmonary dysplasia and pulmonary insufficiency of prematurity. Lack of correlation of outcome with gas exchange abnormalities at 1 month of age.

A review of all infants admitted to the two intensive care nurseries in Seattle from July 1, 1980, through Dec 31, 1981, was performed to evaluate the outcome of infants still requiring supplemental oxygen and/or mechanical ventilation at 1 month of age. Sixty-three infants were identified. Fifty-six infants survived to at least 2 years of age, including 11 of 13 in the subgroup of infants requiring 40% or more oxygen at 1 month of age. Eight (14%) of the 56 survivors have required prolonged rehospitalization for pneumonia or other respiratory illnesses in the first two years following birth. We conclude that the degree of gas exchange impairment assessed at 1 month of age does not predict ultimate outcome from neonatal chronic lung disease.

Evaluation of immunity of sheep after splenectomy, splenic artery ligation and autotransplantation of splenic tissue.

The studies aiming at evaluation of immunity after splenectomy, splenic artery ligation, and autotransplantation of splenic tissue were performed in sheep. It was found that splenectomy alters both humoral and cellular immunity. The phagocytic activity of granulocytes, reduced level of IgG, drop in T and ARF cells as well as bone marrow stimulation were noted. In animals subjected to splenic artery ligation or autotransplantation of splenic tissue no significant differences in parameters studied were observed. These results correlated with histological examinations indicate that in the case of spleen trauma two important steps should be followed: the spleen, if possible, should be saved or a spleen saving procedure should be employed; in case of extensive trauma or if contraindications for spleen saving exist, autotransplantation of splenic tissue should be performed.

Coronal craniostenosis: fetal head constraint as one possible cause.

A retrospective study of 11 instances of idiopathic coronal craniostenosis in otherwise normal children revealed that early lightening, prolonged moderate to severe pelvic discomfort late in pregnancy, and/or an abnormal fetal lie were unusual gestational features indicative of intrauterine constraint for eight of these patients. The impression of unusual constraint in utero was further implied by finding associated positional foot deformities in four of these latter eight children. We hypothesize that prolonged constraint of the fetal head may limit anteroposterior growth stretch at the coronal suture and thereby predispose toward early sutural fusion.

[Late results of surgical development of the artificial vagina from the Douglas's pouch (author's transl)]. / Spätergebnisse nach operativer Bildung einer künstlichen Scheide aus dem Douglasperitoneum.

Late results of surgical development of artificial vagina in 18 patients by the Glowinski method are discussed. The Rokitansky-Küster syndrome was found in 16 women, and pseudomale hermaphrodism in two. In 12 cases the results of operation were positive, satisfactory in 3, and negative in the remaining 3 cases. The surgical method is simple and safe, and from the functional point of view prognosis is favourable.