Isolation, identification & characterization of Proteus penneri - a missed rare pathogen.

Background & objectives: Indole negative Proteus species are invariably incorrectly identified as P. mirabilis, missing isolates of Proteus penneri. P. penneri is an invasive pathogen capable of causing major infectious diseases still seldom reported in individual cases. We report here the isolation, differentiation, characterization and typing of P. penneri from patients with different clinical infections. Methods: Urine, pus and body fluids collected from patients in intensive care units, wards and out patients departments of a tertiary health care institute from north India were cultured. A total of 61 indole negative Proteus isolates were subjected to extended biochemical tests to differentiate and identify P. penneri from P. mirabilis including failure to produce ornithine decarboxylase (by 0% strains of P. penneri and 100% strains of P. mirabilis) besides P. penneri being uniformly salicin negative, non-utilizer of citrate but ferments sucrose and maltose. Antibiograms and Dienes phenomenon were performed to characterize and type P. penneri isolates besides screening for β-lactamase production. Results: Eight isolates of P. penneri were identified; four from urine, three from abdominal drain-fluid and one from diabetic foot ulcer. P. penneri was isolated as the sole pathogen in all patients having underlying disease; post-operatively. Swarming was not seen in the first strain on primary isolation and was poor in strain-4. All eight isolates were biochemically homologous but multi-drug resistant (MDR) with resistance to 6-8 drugs (up to 12). β-lactamase production was seen in three of five isolates while Dienes phenomenon found four distinct types and discriminated strains differing in resistance even with a single drug. Interpretation & Conclusions: A few additional biochemical tests identified P. penneri isolates; it infected patients with underlying disease and strains were MDR and heterogenous.

A pilot study on parvovirus B19 infection in paediatric haematological malignancies.

Background & objectives: Leukaemia and lymphoma are common paediatric haematological malignancies acquiring human parvovirus B19 (B19) infection. In some studies anaemia has been found in children with acute lymphoblastic leukaemia (ALL) during maintenance therapy and rarely in lymphoma. We studied frequency of B19 infection and its implications in new onset acute leukaemia (mostly ALL) and lymphoma in children. Methods: Seventy serum samples from 35 children (age <12 yr, 29 males) newly diagnosed with haematological malignancies (on induction therapy) were collected together with 34 controls (solid tumours). Children were examined clinically and for anti-B19 IgM antibodies by quantitative ELISA and B19 DNA by PCR (VP1-VP2) and nested-PCR (VP1 unique). Bone marrow aspirates were examined histopathologically, whenever possible. Results: Of the 35 children, 22 had acute leukaemia while 13 had lymphoma. B19 infection was seen in six (17.1%) of 35 children (5 ALL, 1 NHL), two at diagnosis and four during follow up compared to none in the control. Among five B19 IgM positive ALL (n=18) children, two had B19 genome and two had giant pronormoblasts (lantern cells; but one lacked B19 DNA). Of the 70 serum samples tested, eight (11.4%) had anti-B19 IgM as two children had persistent B19 infection and one showed atypical maculopapular rashes (lower limbs) while 12 (34.3%) had anti-B19 IgG antibodies. B19 infected children had unexplained anaemia (80%), required more blood transfusions (6.6 ± 4.8 Units vs 3.0 ± 2.6 Units) besides induction chemotherapy was delayed (60%) and required longer duration of therapy (29.2 ± 20 vs 6.3 ± 7.8 days) (P<0.02). Five children (2 ALL, 2 AML, 1 NHL) died but none were infected with B19. Interpretation & conclusions: B19 infection should be considered in children with ALL as it frequently caused unexplained anaemia and delay in induction chemotherapy.

Novel detection of parvovirus B19 DNA & IgM antibodies in patients with non-occlusive gangrene of stomach & bowel.

Background: Gangrene of stomach or intestines owing to non-occlusive bowel infarction (NOBI) is a rare event with unknown etiolology. Since B19 may cause vasculitis, arteritis, angiopathy and more importantly, localized microvascular thrombi formation hence patients with bowel gangrene were investigated for B19 infection. Methods: Twelve patients (8 male and 4 females; median age 40 yr) of ischemic unexplained gangrene of bowel underwent emergency laparotomy. Eight cases had NOBI while four had occlusive bowel infarction (OBI). Anti-B19 antibodies in sera by ELISA and Western-blot and B19 DNA by PCR in sera and resected tissues were analysed. Results: All patients underwent resection of gangrenous bowel; with exteriorization followed by restoration wherever appropriate. Histopathology showed loss of bowel mucosa and crypts with inflammatory cell infiltration besides fibrin thrombus in gastric vessels. Sera of all 8 patents of NOBI had B19 genome by nested-PCR (VP1 unique) and in 6 by PCR (VP1-VP2). In three patients resected bowel tissues also had B19 DNA besides anti-B19 IgM and IgG antibodies. NOBI patients were reticulocytopenic and anaemic while one had necrotizing vasculitis of skin a year ago. No IgM antibodies to agents causing vasculitis (HTLV-I, HIV-1+2, CMV, HSV1+2, mumps virus and Mycobacterium tuberculosis) nor any abnormality in coagulation profiles were detected. In four OBI cases’s sera and resected bowel tissues and in control bowel tissues (n=36) no anti-B19 IgM antibodies or B19 DNA were detected. Conclusion: Novel finding of active B19 infection in non-occlusive gangrene of the bowel may be causal rather than casual.

Prevalence of infections in renal transplant recipients of north India.

Renal transplant is usually performed at the end stage of renal disease. Most of the transplant recipients become susceptible to infections due to chronic uremia, protein depletion, anemia and administration of immunosuppressive drugs. It is a retrospective study of 510 post renal transplant recipients. 378 (74%) renal transplant recipients suffered from the infections. Most common site of infection was urinary tract infection (53%). Out of 26% of wound infections, the deep wound infection (23%) was six times higher than superficial wound infection (3.5%). Chest infection and bacteraemia were noticed to be 18% and 8%, respectively. The common isolate was Escherichia coli (160) followed by Staphylococcus aureus (140), Enterococcus (86) and Pseudomonas (69).

Serological study of parvovirus B19 infection in women with recurrent spontaneous abortions.

The feto-pathogenic association of parvovirus B19 (B19) has been sparingly studied in women with abortion, but not in women with recurrent spontaneous abortions (RSA). Serum samples from 116 women with RSA who were pre-screened for S-TORCH, Chlamydia trachomatis infections, anatomical, chromosomal, endocrinal abnormality and Rh incompatibility and those who had no such known causes of abortion were included in the study. Sera were also collected from 136 normal pregnant women and 120 normal non-pregnant women as disease and normal control respectively. All sera were tested at 1:400 dilutions for B19 IgM by in-house ELISA using cloned and baculovirus expressed VP1 and VP2 antigens of B19. The frequency of anti-B19 IgM antibodies in women with RSA was 19.8%, in pregnant females it was 11% and in normal non-pregnant female was 5%. Sera of 23 women with RSA which were positive for B19 IgM tested negative for B19 DNA by PCR. Patients with RSA should be screened for B19 infection and guidelines for treatment should emerge.

Detection of parvovirus B19 in a case of erythema infectiosum with myositis.

A well documented case of erythema infectiosum is being reported here for the first time from India which was associated with myositis that has not been reported globally. A 9-year-old child presented with moderate to high grade fever, mild anemia, and erythematous rash involving face, trunks and limbs associated with arthralgia, myalgia and myositis. Parvovirus B19 infection was confirmed by detection of IgM antibodies (inhouse ELISA) and DNA (nested PCR) in patient's serum.

Standardization of parvovirus B19 DNA extraction from serum and quantitative PCR.

Human parvovirus B19 (B19) is a newly emerging virus causing a wide spectrum of clinical conditions. The corner stone of diagnosis of acute B19 infection is demonstration of anti-B19 IgM antibodies and its DNA by PCR. Sera of patients infected with B19 acutely or persistently shows intense viraemia (up to 10(12) virus particles/ ml) but extraction of B19 DNA from serum is a problematic task. There is no single standardized, cost-effective in-house method, which can successfully extract DNA of B19 from serum samples and which has been subsequently tested repeatedly by many investigators over time. We describe here an efficient in-house method of extraction of B19 DNA from serum and quantitate extracted DNA by PCR. Firstly, a quantitative PCR was done using 3 microl of a cloned B19 DNA (33.3 pg/ml) mixed in 47 microl of sterile distilled water which were further diluted from 10(-1) up to 10(-7) to find the lower limit of DNA detection. To mimic human serum samples with known quantity of B19, 3 ml of cloned B19 DNA (33.3 pg/ml) were mixed with 47ml of fetal calf serum (FCS; free of B19) and were similarly log diluted from 10(-1) to 10(-7) in 50 ml volume. In-house method of DNA extraction from serum (FCS+B19 DNA) were then performed followed by quantitative PCR. In both the cases, we were able to detect B19 DNA up to 10(-4) dilution which contained 0.6 fg of B19 DNA corresponding to 12 B19 genome equivalents/PCR reaction or 2.4 x 10(2) genome quivalents/ml of serum. Further, the entire procedure was repeated two more times and identical results were obtained confirming its reproducibility. Using this in-house method we extracted and amplified B19 DNA successfully from sera of clinically suspected cases of B19 infection (data not shown). Compared to other studies, the sensitivity of our in-house method was found to be superior hence recommended for developing countries as commercial kits are too costly.

Sero-molecular evaluation of human cytomegalovirus disease in renal transplant rejection.

Cytomegalovirus (CMV) is the most common viral pathogen in renal transplant recipients resulting in graft rejection. The prevalence of CMV disease and renal graft rejection is not well studied in India. Sequential specimens from 32 renal allograft recipients were examined by using CMV IgM specific mu capture ELISA and DNA by PCR. Twelve of the 32 patients were CMV IgM positive and out of 12 patients, 9 had rejection and 4 experienced CMV disease. CMV IgM specific mu capture ELISA helped in diagnosis of CMV disease, though it is less sensitive in detection of rejection. PCR itself was proved not sensitive enough in detecting either CMV disease or rejection. At present, optimal laboratory detection of CMV infection in these patients can be achieved only by multiple and more sensitive parameters.

Neurocysticercosis in clinically suspected and MRI proven cases: evidence of sub-optimal antibody response.

Neurocysticercosis (NCC) has a worldwide distribution mainly in the developing countries like India. The study was done to find the seroprevalence of anti-cysticercus antibodies in clinically suspected and MRI proven cases and to corroborate the serological findings with radiological findings (MRI). A hospital based study among 204 suspected patients during January, 1996 to August, 2001 showed that 77 (32.2%, M:F = 2.2:1) had serological evidence of NCC. Of the total 189 sera, tested at 1:100 dilution 68 (35.9%) and of the total 50 CSF, tested at 1:5 dilution 9 (18%) were positive for anti-cysticercus IgG antibodies. In 35 cases where both were tested 13 sera (37.1%), 9 CSF (25.7%) and in 7 (20%) both sera and CSF were positive. In CSF from 62 patients with tubercular meningitis (disease control) 2 (3.2%) samples whereas in sera of 60 normal blood donors (normal control) 7 (11.7%) samples had anti-cysticercus IgG antibodies. In 33 MRI-positive cases, anti-cysticercus antibodies were seen in 15 (45.4%) patients. Antibodies were seen in 6 of 14 (42.8%) cases with single cortical cyst, 4 of 11 (36.3%) with 2-3 cysts and in 5 of 8 (62.5%) with multiple cysts. Alternatively, 18 of 33 (54.5%) MRI positive cases lacked anti-cysticercus antibodies. Six MRI negative cases were found to be seropositive and were treated successfully. Hence, immune response was sub-optimal even in MRI positive cases and conversely, few MRI negative cases were seropositive. Since positive response with MRI or serology depends on the stage of the disease, therefore both tests should be done together to confirm or to rule out NCC.

Seroanalysis of Chlamydia trachomatis and S-TORCH agents in women with recurrent spontaneous abortions.

Genital tract infections are an important cause of pregnancy loss, many of which are amenable to treatment. There is scarcity of literature on role of S-TORCH agents in recurrent spontaneous abortions (RSA) and available data on role of Chlamydia trachomatis (CT) is controversial. In a retrospective analysis, CT IgM, IgG and IgA antibodies were tested by indirect ELISA in 86, 53 and 30 sera samples respectively from women with RSA (= or > 3 abortions). IgM antibodies using m-capture ELISA for S-TORCH agents (Syphilis, tested by VDRL) were analysed in 47 sera from women with RSA. Results compared with 29 age matched normal pregnant women. Anatomical, endocrine, Rh incompatibility and chromosomal abnormality in the couple were ruled out prior to inclusion in the study. Serum anti-CT IgM positivity was 46.5% in RSA vs. 13.8% in control group (p < 0.001). S-TORCH positivity in RSA group was 31.9% and nil in the control group (p < 0.005). Present study demonstrates a strong association between IgM antibodies to CT and S-TORCH agents in women with history of RSA.

Serodiagnosis of smear and culture-negative neurotuberculosis with enzyme linked immunosorbent assay for anti A-60 immunoglobulins.

Early diagnosis of neurotuberculosis (NTB), useful in prevention of mortality and morbidity, remains a challenge despite availability of several tests. An ELISA test to detect IgG and IgM antibodies against Mycobacterial antigen A-60 (Anda Biologicals, France) was done in 677 specimens; group 1 (NTB): 373 sera and 167 cerebrospinal fluid (CSF), group 2: 100 sera from healthy subjects, group 3: 17 CSF from patients undergoing neurosurgical operations for non-tubercular diseases. Anti-A 60 IgA estimation was done in 99 sera from group 1 and all 100 from group 2. Working dilutions were 1:200 for serum and 1:10 for CSF. Serum IgM and IgG anti-A 60 antibodies were more often detected in group 1 than in 2 (50% Vs 10%, p<.001). Anti-IgG and IgM antibody were detected more often in group 1 than in group 3 (33% Vs 6%, p<.001). In serum and CSF both IgM positivity was more than IgG in 2 subgroups of NTB and these are tubercular meningitis, spinal tuberculosis whereas in tuberculoma IgG positivity was more as compared to other 2 groups. Sera were more often positive than CSF (50% Vs 33%, p<.001). Of 32 patients, in whom magnetic resonance imaging (MRI) was done, 15/18 (83%) patients with suggestive findings in MRI had a positive ELISA (IgG or IgM). AntiA-60 antibody is a useful aid in the diagnosis of NTB, especially in smear and culture negative NTB where one does not have much diagnostic opportunities to choose from.

Detection & differentiation of Coxsackie A 24 variant isolated from an epidemic of acute haemorrhagic conjunctivitis in north India by RT-PCR using a novel primer pair.

BACKGROUND & OBJECTIVES: An epidemic of acute haemorrhagic conjunctivitis (AHC) occurred in north India during July to September 1994. We report a reverse transcription-polymerase chain reaction (RT-PCR) using known and novel primers to differentiate and identify the CA 24 virus isolated from the epidemic of AHC. METHODS: Conjunctival swabs were collected from 46 patients (in 12 patients from both the eyes) yielding 58 swabs. The swabs were inoculated in RD 19S and HeLa-199 cell monolayers and observed for cytopathic effect. Serum neutralizing antibodies were tested in 17 acute and 10 convalescent phase serum samples. RT-PCR was done on 9 isolates (7 Coxsackie A 24 and 2 ECHO-1 as identified by neutralization test) using known and a novel primer. Fourteen virus isolates (9 CA 24, 3 ECHO-1 and 2 untyped) were inoculated in suckling mice and these mice were observed daily for 10 days for flaccid paralysis of hind limb or death. RESULTS: Cytopathic virus was isolated from conjunctival swabs in 21 of 46 (45.6%) patients subjected to virus isolation. Sixteen of 21 (76.2%) isolates were neutralized by CA 24 specific antisera, 3 isolates were identified as ECHO-1 with Schmidt enteroviruses antiserum pools while 2 remained untypable. Of these 21 isolates, 9 representative isolates (7 CA 24 and 2 ECHO-1) tested by RT-PCR had enterovirus common region DNA but did not show any amplification in RT-PCR with EV-70 specific primers (VP-1 and VP-3). Using CA 24 specific novel (VP 3-1) primers amplification was seen in 6 of 7 CA 24 isolates while 2 ECHO-1 remained unamplified. In contrast with 3C-proteinase region primers, only 2 of 7 CA 24 were amplified along with false amplification of both ECHO-1. Serum neutralizing antibodies were seen in 2 of 17 (11.7%) acute phase sera and 6 of the 10 (60%) convalescent phase sera while in paired sera (available in two patients) a four-fold rise in titres were observed. Hind-limb paralysis and/or death occurred in all suckling mice inoculated with CA 24 isolates while mice remained healthy after inoculation with 3 isolates of ECHO-1 and 2 untypable isolates. INTERPRETATION & CONCLUSION: The epidemic of AHC was caused by a variant of CA 24. Molecular typing can detect and differentiate between CA 24 and EV-70 viruses. Novel primer pair was found useful in the identification and confirmation of CA 24 isolates.