[Continuous axenic cultivation of Pneumocystis carinii isolated from the bronchoalveolar lavage fluid of infected rat].

OBJECTIVE: To establish axenic cultivation of Pneumocystis carinii (P.c). METHODS: The organisms of P.c were isolated from the bronchoalveolar lavage fluid (BALF) of the rats with Pneumocystis carinii pneumonia (PCP) and cultured in a medium which was based on IMDM(GIBCO) supplemented with S-adenosyl-L-methionine, putrescine, N-acetyl glucosamine, putrescine, L-cysteine and L-glutamine, and newborn calf serum. The organisms cultured in the system were identified by observing the morphology of cysts in smears stained with Gomori's methenamine silver nitrate stain (GMS). Ultrastructure of the cysts/trophozoites was examined by transmission electron microscopy. The sequences of mitochondrial large ribosomal DNA subunit of the cultured organisms were compared with the Pneumocystis carinii f.sp. ratti variant isolate (GenBank No U20173) and Pneumocystis carinii f.sp.hominis (GenBank No M58605). RESULTS: Five isolates of P. carinii received from BALF of 8 rats with PCP were cultured axenically and continuously in the system. The cultured organisms could be stored in frozen condition and used to reinitiate culture, and were amplified by 19-22 times within 72 h. The morphology, ultrastructure and gene sequencing of the cultured organisms confirmed that the isolated organisms were P. carinii. CONCLUSION: Five continuously and axenicly cultured isolates of P. carinii have been received.

[Clinical observation on 25 cases of severe angiostrongyliasis cantonensis].

OBJECTIVE: To summarize the clinical features of severe angiostrongyliasis cantonensis (AC) patients. METHODS: Clinical data on symptoms, physical signs, auxiliary examination and prognosis of 25 severe AC patients hospitalized in June-Sept 2006 were analyzed. RESULTS: Epidemiologically, all cases had eaten uncooked fresh water snails. One of the early symptoms was fever (16 cases, 64.0%), including 8 cases with low-grade fever, 7 cases with mid-range fever, and 1 case high fever. Nervous system manifestation: (1) All cases had headache, entire headache (56.0%) or partial (44.0%), especially in occipitalis. (2) Patients had distinct degree neck rigidity, with negative pathologic reflex; 12 cases had nausea and vomiting (48.0%). (3) 20 cases (80.0%) had skin paresthesia, 5 had severe pain and hyperalgia on skin; 3 cases with skin numbness, and 2 with thermohypesthesia. (4) 11 cases (44.0%) appear distinct degree depraved vision; 3 cases had photophobia, 5 with blur vision, 1 each with diplopia, defect of field vision or bug sign, respectively. (5) Nasolabial groove became shallow and distortion of commissure in 4 cases (16.0%), and 2 cases (8.0%) couldn't close up eyelid. (6) 4 cases had sustained or curative tinnitus. Laboratory examination showed that eosinophilic granulocytes increased in both peripheral blood and cerebrospinal fluid. Skull MRI for 14 cases revealed linear enhancement in local meninx or abnormal enhancement in cerebral parenchyma. Chest CT examination in 7 cases showed nodule shadow and spot flaky ground-glass shadow in lungs. One and 3 months after being discharged from hospital, 12 patients (48.0%) still had sequelaes--7 cases had tingling sensation on skin, 1 case had temperature sensation dysfunction on the skin of chest and abdomen, 3 cases had headache occasionally, and 1 case still had defect of field vision. CONCLUSION: Central nervous system has been impaired in the angiostrongyliasis cantonensis patients who may need a longer convalescent period.

Genotyping of Pneumocystis jirovecii isolates from human immunodeficiency virus-negative patients in China.

Pneumocystis jirovecii is a fungus that causes Pneumocystis pneumonia in immuno-compromised patients. To analyze the genetic diversity of P. jirovecii in HIV-negative patients in China, respiratory specimens were obtained from 105 patients who tested PCR-positive for the presence of the P. jirovecii mitochondrial large subunit ribosomal RNA (mtLSU rRNA) between 2011 and 2013. P. jirovecii isolates were genotyped based on the upstream conserved sequence (UCS) of the major surface glycoprotein (MSG) gene and the internal transcribed spacer (ITS) of nuclear rRNA operon. Eighty-one of the 105 isolates showed a positive PCR for the UCS region. We identified six different patterns comprising two, three, four, or five UCS repeats, including 1, 2, 3 (69.14%), 1, 2, 3, 3 (22.22%), 1, 2 (3.7%), 1, 1, (2.47%), 2, 2, 3, 3 (1.23%), and 1, 1, 2, 3, 3 (1.23%). In regard to the ITS region, 58 of the 105 isolates were cloned and sequenced successfully. Six known ITS1 alleles (A, B, DEL1, E, N, and SYD1), two new alleles (designated as BTM3 and BTM4), six known ITS2 alleles (a, b, i, g, h and O) and one new allele (designated as btm6) were observed. A total of 19 P. jirovecii ITS haplotypes were identified. The most frequent type was Bi (25.9%), followed by Ai (13.8%), Eb (10.3%), and SYD1g (6.9%). Among the 58 specimens examined, 49 (84.5%) were found to contain a single type of P. jirovecii, while 9 (15.5%) contained multiple genotypes. A total of 34 allelic profiles were observed in 58 isolates when the two loci were combined with each other. A Fisher's exact test revealed that there was no statistically significant (P=0.330) association between the most frequent UCS and ITS genotypes. An analysis of the phylogenetic relationship between different patient groups identified two major groups based on the sequence variations of concatenated UCS and ITS sequences in 49 isolates. Our results demonstrated the high genetic variability of P. jirovecii in HIV-negative patients in China.

[An epidemiologic study on Pneumocystis pneumonia in non-HIV infected patients in China].

OBJECTIVE: To analyze the epidemiologic features of Pneumocystis pneumonia (PCP) among non-HIV infected patients in China. METHODS: Sputum or bronchoalveolar lavage fluid (BALF) specimens obtained from 851 pneumonia patients without HIV infection from Jan. 2006 to Oct. 2008 were detected, using PCR and Gomori' s methenamine silver (UMS) stain for Pneumocystis jirovecii. RESULTS: Of the 615 sputum specimens, P. jirovecii positive rates of PCR and GMS stain were 20.3% and 10.2% respectively (P < 0.05). Of 236 BALF specimens, P. jirovecii positive rates of PCR and GMS stain were 32.6% and 25.5% respectively (P > 0.05). Of the total 851 pneumonia cases, 123 (14.5%) were GMS positive for P. jirovecii cyst and 202 cases (23.7%) were PCR positive for P. jirovecii DNA. In those immuno-suppressed patient group including patients with connective tissue diseases, organ transplant recipients, nephrotic, hematologic diseases and malignant tumor, P. jirovecii positive rate appeared the highest, 28.2% for GMS stain and 39.4% for PCR. There were also PCP patients in the immuno-competent pneumonia patient groups including senile patients with chronic diseases and patients without clear predisposing immuno-deficiencies. The positive rates of P. jirovecii GMS were 8.7% and 10.9%, respectively and 17.5% and 19.6% for P. jirovecii under PCR. CONCLUSION: PCR assay seemed sensitive for the detection of P. jirovecii in the sputum specimens and could be used for screening PCP patients without HIV infection. Our data showed that there was high risk of PCP in non-HIV infected patients in China.

[Epidemiological analysis on 141 cases of angiostrongyliasis cantonensis in Beijing].

OBJECTIVE: To provide scientific basis for angiostrongyliasis cantonensis control and prevention in Beijing. METHODS: Descriptive epidemiological method was used to analyze angiostrongyliasis cantonensis reported from June to September in 2006. RESULTS: 141 cases were treated at the Beijing Friendship Hospital with the peak in July (61 cases) and August (68 cases). All patients had dined at the same restaurant in Beijing city and they ate undercooked Pomacea canaliculata or related eatables. The source of Pomacea canaliculata was from Guilin in Guangxi. Major manifestations would include fever (56.79%), headache (93.83%), neck stiffness (100%), and skin paresthesia (77.78%). Some cases had significant eosinophil increase in peripheral blood picture and cerebrospinal fluid respectively. CONCLUSION: The source of infection related to angiostrongyliasis cantonensis was clear, suggesting that the improvement of restaurant sanitation and on awareness of personal hygiene were important preventive and control measures on angiostrongyliasis cantonensis.