Clinical and Microbiological Characteristics of Bacteremia Caused by Carbapenemase-producing Enterobacterales in Minami Ibaraki Area, Japan.

Although recent propagation of carbapenemase-producing Enterobacterales (CPE) has become a problem worldwide, the picture of CPE infection in Japan has not fully been elucidated. In this study, we examined clinical and microbiological characteristics of invasive CPE infection occurring at 8 hospitals in Minami Ibaraki Area between July 2001 to June 2017. Of 7294 Enterobacterales strains isolated from independent cases of bacteremia and/or meningitis, 10 (0.14%) were CPE (8 Enterobacter cloacae-complex, 1 Escherichia coli, and 1 Edwardsiella tarda）, all of which had the blaIMP-1 gene and susceptible to gentamicin and trimethoprim/sulfamethoxazole. These strains were isolated from 7 adult and 2 infant bacteremia (1 infant patient developed CPE bacteremia twice) after 2007. The most common portal of entry was intravenous catheters. All of the adult patients were recovered, while the infant patients eventually died. Genomic analyses showed that the 8 E. cloacae-complex strains were classified into 5 groups, each of which was exclusively detected in specific facilities at intervals of up to 3 years, suggesting persistent colonization in the facilities. This study showed that invasive CPE infection in the area was rare, caused by IMP-1-type CPE having susceptibility to various antibiotics, and nonfatal among adult patients.

Solitary pulmonary caseating granulomas: A 5-year retrospective single-center analysis.

Solitary pulmonary caseating granulomas (SPCGs) are a characteristic type of tuberculomas associated with infection with non-tuberculous mycobacteria (NTM) and other microbes; however, their significance remains unclear. The aim of the present study was to describe the clinical characteristics of patients with SPCGs in terms of diagnosis, presence of lung cancer and treatment status. A retrospective analysis of 17 immunocompetent patients with histopathologically confirmed caseating granulomas after undergoing video-assisted thoracoscopic surgery (VATS) was conducted at our center between 2011 and 2015. The patients comprised 10 men and 7 women with a mean age of 59.1±14.4 years. Of the 17 patients, 14 (82.4%) were asymptomatic and the lesions were discovered incidentally. In 2 patients the SPCGs were accompanied by a small satellite nodule (SPCG mean diameter, 16.2±5.1 mm). Mycobacteria, including Mycobacterium tuberculosis (11.8%), Mycobacterium avium (11.8%) Mycobacterium kansasii (23.5%) and other Mycobacterium spp. (5.9%), were isolated from 9 of the patients (52.9%). Concurrent lung cancer was present in 3 patients (17.6%). When microbial agents could not be isolated, the interferon-γ release assay was useful for diagnosis. Positron emission tomography was not found to be useful for differentiating SPCGs from lung cancer, or for differentiating tuberculomas from NTM pulmonary nodules (NTMPNs). NTMPNs in cases of SPCGs were diagnosed more frequently in men. The findings indicate that a course of observation may be sufficient for patients in whom an SPCG from NTM (NTMPN) is identified by VATS. However, the presence of concurrent lung cancer in certain cases indicates that malignancy should not necessarily be excluded, particularly in NTMPNs, and highlights the necessity of aggressive diagnosis by VATS.

Successful desensitization therapy involving fluoroquinolone for the treatment of a solitary tuberculoma: A case report and literature review.

The patient was a 31-year-old female with no previous health problems; however, during a health checkup in 2013, a nodule (2.5 cm in diameter) was identified in the S10 area of the left lung. No clinical symptoms were apparent. Positron emission tomography/computed tomography revealed an accumulation in the same region. The patient was suspected of having lung cancer, and video-assisted thoracoscopic surgery was performed. A histopathological examination of the resected specimen revealed epithelioid granulomas accompanied by caseous necrosis in the lesion. The culture was positive for Mycobacterium tuberculosis, which led to the final diagnosis of tuberculoma. Initially, the patient underwent anti-M.tuberculosis treatment [isoniazid (INH) + rifampicin (RFP) + ethambutol (EB) + pyrazinamide (PZA)]. However, two weeks later, the development of epatic dysfunction necessitated suspension of the medication. Treatment was resumed following improvement of the hepatic function. However, this relapsed two weeks later, resulting in discontinuation of the treatment. The patient was negative for each of the four drugs in the drug-induced lymphocyte stimulation test (DLST), and drug-induced hepatotoxicity (DIH) attributable to the anti-tuberculous drugs that were administered. Therefore, desensitization therapy was initiated. EB + PZA were changed to levofloxacin (LVFX) at an initial dose of 250 mg/day (dose level increased to the maintenance dose). Subsequently, desensitization therapy with RFP and INH was applied in accordance with the Japanese Society for Tuberculosis protocol. After each drug dose level reached the maintenance dose level, the therapy was completed following administration of the drugs for the recommended duration of 6 months. There were no signs of relapse 6 months following completion of the therapy. Therefore, the patient responded well to the substitute therapy with LVFX and desensitization therapy, and the present case report provided information regarding the treatment of tuberculoma.

High-level aminoglycoside resistance in Enterococcus faecalis and Enterococcus faecium causing invasive infection: Twelve-year surveillance in the Minami Ibaraki Area.

We examined prevalence of high-level aminoglycoside resistance (HLAR) in Enterococcus faecalis and Enterococcus faecium causing invasive infection in the Minami Ibaraki Area. Ten strains of both species each, recovered from the blood or the cerebrospinal fluid between 2003 and 2014, were randomly selected every year. High-level resistance to gentamicin (HLR-GM) and streptomycin (HLR-SM) was detected in 34% (41 of 120 strains) and 18% (21) of E. faecalis and 9% (11) and 39% (48) of E. faecium, respectively. In comparisons of the proportions among three four-year periods, HLR-SM among E. faecium was significantly lower in the 2011-2014 period. All strains with HLR-GM were positive for the aac(6')-Ie-aph(2″)-Ia gene. The ant(6')-Ia gene was detected in all with HLR-SM except for one E. faecalis strain. The present study showed that prevalence of HLR-GM among E. faecalis and E. faecium causing invasive infection in this area was nearly equivalent to that described in previous studies in Japan and that proportions of strains with HLAR did not vary during the study period except for that of HLR-SM among E. faecium.

Characterization of a human isolate of Tritrichomonas foetus (cattle/swine genotype) infected by a zoonotic opportunistic infection.

Tritrichomonas species flagellates (IMC strain) were isolated from the biliary tract of an individual who had developed cholecystitis as a complication of acquired agammaglobulinemia. Sequence analysis of Tritrichomonas sp. (IMC clone 2 (cl2)) was performed for several genetic regions including the ITS1-5.8S rDNA-ITS2 region, the cysteine protease (CP)-1, CP-2 and CP-4 to CP-9 genes, and the cytosolic malate dehydrogenase 1 gene. In addition to comparison of the variable-length DNA repeats in the isolate clone with those in T. foetus (Inui cl2) and the T. mobilensis (U.S.A.: M776 cl2) reference strains, this analysis showed that the Tritrichomonas sp. (IMC cl2) was T. foetus (cattle/swine genotype). Injection of T. foetus (IMC cl2) directly into the livers of CBA mice resulted in liver abscess formation on Day 7. Moreover, inoculation via orogastric intubation caused infection in the cecum on Day 5 in CBA mice co-infected with Entamoeba histolytica (HM-1: IMSS cl6). T. foetus (IMC cl2) was able to grow in YI-S medium for over 20 days, even at 5°C. These results indicate that the T. foetus isolate is able to survive in the feces and edible organ meat of the definitive host for a prolonged period of time, and it is possible that the parasite could infect humans.

Greater Cervical Muscle Fat Infiltration Evaluated by Magnetic Resonance Imaging is Associated With Poor Postural Stability in Patients With Cervical Spondylotic Radiculopathy.

STUDY DESIGN: A population-based, cross-sectional study. OBJECTIVE: The aim of this study was to quantitatively evaluate the relationship between static postural stability and fat infiltration within cervical multifidus muscle in patients with cervical spondylotic radiculopathy (CSR). SUMMARY OF BACKGROUND DATA: CSR causes denervation by compression of nerve roots. This denervation is detected by fatty infiltration or results in fatty infiltration within muscles. Proprioceptive information in cervical multifidus muscle plays an important role in coordinated movement of postural stability; however, there have been few studies evaluating the relationship between postural stability and fat infiltration within cervical multifidus muscle among CSR patients. METHODS: Sixteen CSR patients with C6 injuries and 25 age-matched healthy controls underwent magnetic resonance images to examine bilateral cervical multifidus muscle. For evaluation of fat within muscle, a muscle fat index (MFI) was calculated by using both measurement of cervical multifidus muscle and intermuscular fat. Participants' postural stability at upright position with eyes-opened and eyes-closed for 60 seconds was examined by a platform. Two parameters, the total length and the area of the center of pressure (COP), were used for evaluation. RESULTS: The CSR group showed significantly poorer postural stability than the control group (eyes-opened the total length; P < 0.05, eyes-closed the total length; P < 0.05, eyes-closed the area; P < 0.05). There were significant group differences at C4, C5, and C6 MFI (P < 0.05). In the CSR group, a correlation analysis demonstrated that the age, C4, C5, and C6 MFI values were significantly associated with the eyes-closed the total length of the COP (P < 0.05). CONCLUSIONS: Fat infiltration within muscle could lead to inhibition of normal activity of musculature. The present study suggests that fat within cervical multifidus muscle could directly cause postural instability in static standing, even though the proprioceptive information has normal lower limbs.

Native valve endocarditis caused by Neisseria elongata Subsp. nitroreducens.

Neisseria elongata, a normal resident in the human oral cavity, rarely causes invasive infections. We herein report a case of endocarditis caused by Neisseria elongata subsp. nitroreducens that occurred in a patient without any apparent cardiac complications. The patient received aortic valve replacement following the administration of intravenous beta-lactam for five weeks. To our knowledge, this is the first published case in Japan of N. elongata infection in a patient without a prosthetic device.

Central nervous system infection caused by vancomycin-intermediate Staphylococcus aureus (SCCmec type IV, ST8).

A 77-year-old Japanese man with a history of surgical treatment of chronic subdural hemorrhage was hospitalized for drainage of a subdural abscess and brain abscess in the right occipital area. Pus obtained from both the subdural abscess and brain abscess grew vancomycin-intermediate Staphylococcus aureus (VISA) (minimum inhibitory concentration = 4 µg/mL), which was confirmed by population analysis. The SCCmec type and sequence type were subsequently identified as IV and ST8, respectively. The VISA strains were both sensitive to levofloxacin, clindamycin, minocycline, and linezolid. The patient was successfully treated with linezolid and discharged on day 51 after admission. We herein describe the first reported case of a brain abscess and subdural abscess caused by VISA in Japan.

Characteristics of bacteremia caused by extended-spectrum beta-lactamase-producing Proteus mirabilis.

Although Proteus mirabilis is a common human pathogen, bacteremia caused by the organism, especially strains producing extended-spectrum beta-lactamase (ESBL), has rarely been investigated. We examined 64 cases of P. mirabilis bacteremia identified in the Minami Ibaraki Area, Japan, between 2001 and 2010 and compared the characteristics of cases with ESBL-producing and ESBL-non-producing strains (13 and 51 cases, respectively). All ESBL-producing strains with the gene encoding the CTX-M-2-group were genetically nonidentical. Isolation of ESBL-producing strains was significantly associated with onset in a hospital (p = 0.030), receiving hemodialysis (p = 0.0050), and previous antibiotic use within 1 month (p = 0.036; especially penicillin and/or cephalosporin (p = 0.010) and fluoroquinolone (p = 0.0069)). Isolation was also associated with inappropriate antibiotic therapy on the 1st and 4th days (p = 0.011 and 0.032, respectively) but not with mortality on the 30th day. These findings indicate that, for P. mirabilis bacteremia, isolation of ESBL-producing strains causes delay of initiating appropriate antimicrobial therapy but may not be associated with mortality.

Analysis of the influence of drug resistance factors on the efficacy of combinations of antibiotics for multidrug-resistant Pseudomonas aeruginosa isolated from hospitals located in the suburbs of Kanto area, Japan.

Infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa are very difficult to treat. The aim of this study was to develop more effective treatments by investigating in vitro the effects of combinations of antibiotics against 47 MDR P. aeruginosa isolates harbouring various resistance factors. The isolates included 41 (87%) metallo-ß-lactamase (MBL)-positive strains, 37 (79%) strains with mutations in OprD and 46 (98%) strains carrying the genes encoding aminoglycoside-modifying enzymes (AMEs). The quinolone resistance-determining region was mutated in all of the strains. These strains were classified into 16 groups according to amplified fragment length polymorphism and resistance factors. The effects of combinations of antibiotics on 16 representative strains were determined using a 'Break-point Checkerboard Plate' assay. Combinations of amikacin+aztreonam (coverage rate, 81.3%) and arbekacin+aztreonam (93.8%) inhibited growth. In contrast, combinations of ciprofloxacin+meropenem (6.3%) and ciprofloxacin+ceftazidime (12.5%) were much less effective. Aztreonam and arbekacin (or amikacin) are not substrates for MBLs and AMEs, respectively. We conclude that the combined effects of these drugs were possibly because of resistance factors.

In vitro combination effects of aztreonam and aminoglycoside against multidrug-resistant Pseudomonas aeruginosa in Japan.

The aim of this study was to evaluate the in vitro combination effects of aztreonam (AZT) and aminoglycosides against multidrug-resistant (MDR) Pseudomonas aeruginosa strains in Japan. We investigated 47 MDR P. aeruginosa strains collected from 8 facilities. We selected the aminoglycosides amikacin (AMK), gentamicin (GM), and arbekacin (ABK) to examine their effects when combined with AZT using the checkerboard method. Of the 47 MDR P. aeruginosa strains, 41 tested positive for metallo-ß-lactamase (MBL). In all combinations, aminoglycosides decreased the minimum inhibitory concentrations of AZT in a dose-dependent manner, and there was no apparent antagonism. The combination effects were scored on a scale of 0 to 4, and statistical analysis was performed using the Wilcoxon signed-rank test. In all 47 strains, AZT + ABK (mean score, 2.02) had the highest score, followed by AZT + AMK (1.68) and AZT + GM (1.38) (ABK versus GM, P < 0.0001). In 41 MBL-positive strains, AZT + ABK (mean score, 2.05) had the highest score, followed by AZT + AMK (1.56) and AZT + GM (1.37) (ABK versus AMK, P = 0.02, and ABK versus GM, P < 0.0001). AZT + ABK was the most promising combination regimen against MDR P. aeruginosa strains; the other promising combinations were AZT + AMK and AZT + GM.

[Urgent hemostatic control with Confact F for ovarian bleeding complicated by an unknown type of von Willebrand disease].

Emergency surgery was performed on a 24-year-old female patient with an unknown type of von Willebrand disease (VWD). The patient suddenly developed lower abdominal pain and was transported to our hospital by ambulance. Based on the diagnosis of ovarian bleeding, emergency surgery was indicated. Ristocetin-cofactor activity (VWF: RCo) was between 7% and 14% by measurement with von Willebrand reagent (Dade Behring, Marburg, Germany). A laparoscopic partial resection of the left ovary was performed under administration of factor VIII/VWF concentrate, Confact F((R)) (Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan). During and after surgery, there was no abnormal bleeding. The results obtained by measurement by the reagent were similar to the levels of VWF: RCo obtained by the fixed platelet agglutination method. The reagent was useful for diagnosing and monitoring the VWF: RCo level to control bleeding in VWD.

[Comparison of antimicrobial use density (AUD) of carbapenem antibacterial agents and investigation of the drug susceptibility of Pseudomonas aeruginosa in 3 hospitals in southern Ibaraki Prefecture, Japan].

The optimal use of anti-Pseudomonas agents is an important issue in the prevention of a tolerance against Pseudomonas aeruginosa. We evaluated the effect of antimicrobial use density (AUD) of carbapenem on drug susceptibility. The AUD of the four carbapenems, imipenem (IPM/CS), panipenem (PAPM/BP), meropenem (MEPM), and biapenem (BIPM), was examined at three hospitals in Ibaraki Prefecture, between April and September 2004. The AUD was calculated using the Defined Daily Doses (DDD) methodology developed by the WHO. A drug susceptibility test was conducted on the 306 Pseudomonas aeruginosa strains randomly collected from clinical specimens at the three hospitals between September and December 2004. In accordance with the standards set by the Clinical and Laboratory Standards Institute (CLSI), minimal inhibitory concentration (MIC) was measured using the broth microdilution method. The results showed that the AUD of carbapenem at the three hospitals tended to be higher than that in other research results in Japan. At one of the three hospitals, the AUD of the PAPM was remarkably high compared to the other carbapenems. Furthermore, P. aeruginosa strains collected at this hospital showed a low susceptibility to carbapenem, and many highly tolerant strains were also observed in this hospital. In order to maintain the susceptibility of Pseudomonas aeruginosa to carbapenem, the overall extent of carbapenem use must be optimal. The use of antimicrobial drugs should be controlled properly at each hospital, in order to prevent excessive use of PAPM/BP from being used over a long period of time.

[Precautions regarding prevent acute urethritis caused by Neisseria meningitidis in Japan].

In Japan, Neisseria meningitidis is not sufficiently recognized as the primary causative bacteria of sexually transmitted diseases (STDs) as the number of reported cases is small. Here, we summarize reports from 3 medical institutions, present clinical courses for each case, as well recommending precautions to prevent infection with this bacterium. Fourteen cases of N. meningitidis urethritis (MU) were admitted between April 2001 and June 2006. All patients were male, consulted a doctor after experiencing subjective symptoms, such as micturition pain and pus discharge, and were diagnosed as having urethritis using isolation culture methods. In 8 of the 14 cases, history of sexual contact in the preclinical stage was confirmed, and contact was with a commercial sex worker (CSW) in 6 of these cases. Many of these patients recalled oral contact. All strains indicated susceptibility to many drugs, and there were no problems with treatment. With regard to serotype, there were 10 cases of type Y, 1 case of type B, and 3 cases that were not classifiable or unidentified. In addition, among the 9 strains that were subjected to genotype identification, 7 strains were ST-23. The recent increase in availability of nucleic acid amplification methods has facilitated simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis. However, we fear that MU will become latent. For screening of urethritis, Gram staining and culture of urethral material must be performed to detect this disease. The relationship of the detected strain and its role in the pathogenesis of meningitis are uncertain, but its serotype and genotype are common in cases of meningitis. Thus, precautions are required to prevent spread of this bacterium.

Thrombus and encapsulated hematoma in cerebral cavernous malformations.

Thrombi, encapsulated hematomas, and granulation tissue are frequently seen in cerebral cavernous malformations (CCMs). We investigated the role that these histological changes play in repeated hemorrhages in CCMs as well as lesion growth, examining specimens of CCMs surgically harvested from 20 patients. The immunohistochemical study included thrombomodulin (TM) and endothelial cell protein C receptor (EPCR), which are important regulators of blood coagulation. Thick capsules, which contained blood degradation product, were seen in cases with encapsulated hematomas. Clusters of sinusoidal vessels were found outside of these thick capsules. Granulation tissue with inflammatory infiltrates and capillaries was seen in 4 cases with non-capsulated hematomas. Organizing thrombi were seen in sinusoidal vessels in 15 out of 20 cases. Factor VIII-related antigen staining demonstrated numerous capillaries in and around organizing thrombi and within the thickened vessel walls as well as in both the inner and outer sides of the hematoma capsule. TM and EPCR were positive in the endothelial cells of these capillaries, whereas they were negative in those of capillaries in the brain surrounding the lesions. Our study suggests that thrombosed sinusoidal blood vessels could gradually expand by repeated bleeding from numerous capillaries inside the wall and become encapsulated hematomas, and capillaries outside the thickened vessel wall could become sinusoidal blood vessels. Thrombosis within cerebral venules could be one of the causal factors of CCMs.

Detrended fluctuation analysis of genome-wide copy number profiles of glioblastomas using array-based comparative genomic hybridization.

We examined whole genomic aberrations of biopsied samples from 19 independent glioblastomas by array-based comparative genomic hybridization analysis. The highest frequencies of copy number gains were observed on RFC2 (73.3%), EGFR (63.2%), and FGR, ELN, CDKN1C , FES, TOP2A, and ARSA (57.9% each). The highest frequencies of copy number losses were detected on TBR1 (52.6%), BMI1 (52.6%), EGR2 (47.4%), DMBT1 (47.4%), MTAP (42.1%), and FGFR2 (42.1%). The copy number gains of CDKN1C and INS and the copy number losses of TBR1 were significantly correlated with longer survival of patients. High-level amplifications were identified on EGFR, SAS/CDK4, PDGFRA, MDM2, and ARSA. These genes are assumed to be involved in tumorigenesis or progression of glioblastomas. The first attempts to apply detrended fluctuation analysis to copy number profiles by considering the reading direction as the time axis demonstrated that higher long-term fractal scaling exponents (alpha2) correlated well with longer survival of glioblastoma patients. The present study indicates that array-based comparative genomic hybridization analysis has great potential for assessment of copy number changes and altered chromosomal regions of brain tumors. Furthermore, we show that nonlinear analysis methods of whole genome copy number profiles may provide prognostic information about glioblastoma patients.

Combined proteomic approach with SELDI-TOF-MS and peptide mass fingerprinting identified the rapid increase of monomeric transthyretin in rat cerebrospinal fluid after transient focal cerebral ischemia.

Several proteins are known to be markedly expressed in the brain during cerebral ischemia, however the change in protein profiles within the cerebrospinal fluid (CSF) after an ischemic insult has not been fully elucidated. We studied the changes in the CSF proteome in rat transient middle cerebral artery occlusion model. Surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS) was used to detect the time-course changes in CSF protein patterns after transient focal brain ischemia. According to hierarchical cluster analysis by self-organising tree algorism (SOTA), the temporal pattern of protein peaks was divided into four groups: acute increase group, chronic increase group, gradual decrease group and unchanged group. In the acute increase group, the expression of a 13.6-kDa protein was markedly increased during the acute phase. The 13.6-kDa protein was identified as monomeric form of transthyretin using two-dimensional electrophoresis and peptide mass fingerprinting based on matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The monomeric transthyretin may represent an ischemia-specific CSF marker to indicate the sequential changes according to ischemic insults of the brain.

Surveillance of vancomycin-resistant Enterococcus in hospitals in Ibaraki prefecture.

We surveyed the prevalence of vancomycin-resistant Enterococcus (VRE) in six hospitals in Ibaraki prefecture. Three hundred and fifty-five fecal specimens were examined and 18 Enterococcus intermediately resistant to vancomycin were isolated. All of them were vanC genotypes (16 vanC-1 and 2 vanC-2 genotypes) and susceptible or intermediately resistant to ampicillin, teicoplanin, linezolid, and quinupristin-dalfopristin. Neither the vanA nor vanB gene was detected. Two of the vanC genotypes were not motile. We concluded that VRE of either VanA or VanB phenotype or those highly resistant to the antibiotics commonly used against enterococcal infection were not epidemic in this prefecture to date. In addition, we consider that detection of vancomycin-resistant genes should be encouraged for the characterization of VRE, because the vanC genotypes are occasionally motility-negative and can be misinterpreted as other Enterococcus species.

[Computer-aided surface area measurement of temporal bone pneumatization: histological sections].

Contention exists on the development of pneumatization of temporal bone. Many techniques to measure the volume of pneumatization have been reported, but no techniques for direct surface area measurement. We measured the surface area and volume of human mastoid air cells. Eight normal temporal bones removed at autopsy were analyzed and fixed in formalin fixative, decalcified, and embedded in celloidin, sectioned at 25 microns and stained with H-E for histological examination. Total surface area and the volume of human mastoid air cells were measured using a personal computer. The surface area of pneumatic spaces for the 8 temporal bone specimens ranged from 36.1 cm2 to 163.0 cm2 (mean +/- SD: 89.1 +/- 34.0 cm2). The volume of pneumatic spaces for the 8 specimens ranged from 1.53 ml to 6.03 ml (mean +/- SD: 4.12 +/- 0.97 ml). The surface area of temporal bone pneumatic spaces we determined could serve as useful basic data for determining the physiology of ventilation for the temporal bone and the function of mastoid cells.

Interference between D30N and L90M in selection and development of protease inhibitor-resistant human immunodeficiency virus type 1.

We studied the evolutionary relationships between the two protease inhibitor (PI) resistance mutations, D30N and L90M, of human immunodeficiency virus type 1 (HIV-1). The former is highly specific for nelfinavir resistance, while the latter is associated with resistance to several PIs, including nelfinavir. Among patients with nelfinavir treatment failure, we found that D30N acquisition was strongly suppressed when L90M preexisted. Thus, D30N/L90M double mutations not only were detected in a very limited number of patients but also accounted for a minor fraction within each patient. In the disease course, the D30N and L90M clones readily evolved independently of each other, and later the D30N/L90M double mutants emerged. The double mutants appeared to originate from the D30N lineage but not from the L90M lineage, or were strongly associated with the former. However, their evolutionary pathways appeared to be highly complex and to still have something in common, as they always contained several additional polymorphisms, including L63P and N88D, as common signatures. These results suggest that D30N and L90M are mutually exclusive during the evolutionary process. Supporting this notion, the D30N/L90M mutation was also quite rare in a large clinical database. Recombinant viruses with the relevant mutations were generated and compared for the ability to process p55gag and p160pol precursor proteins as well as for their infectivity. L90M caused little impairment of the cleavage activities, but D30N was detrimental, although significant residual activity was observed. In contrast, D30N/L90M demonstrated severe impairment. Thus, the concept of mutual antagonism of the two mutations was substantiated biochemically and functionally.