Differential diagnosis of scrub typhus meningitis from bacterial meningitis using clinical and laboratory features.

BACKGROUND: Central nervous system (CNS) involvement in the form of meningitis or meningoencephalitis is common in scrub typhus. As specific laboratory methods remain inadequate or inaccessible in developing countries, prompt diagnosis is often difficult. AIM: To identify the clinical and laboratory parameters that may help in differentiating scrub typhus meningitis from bacterial meningitis. SETTING AND DESIGN: This is a cross-sectional analysis of adult patients admitted with scrub typhus and bacterial meningitis to a tertiary care teaching institute in South India. MATERIALS AND METHODS: A comparison of clinical and laboratory features of 25 patients admitted with meningitis to a university teaching hospital during a 15-month period was made. These patients had meningitis diagnosed based on abnormal cerebrospinal fluid (CSF) analysis with either positive IgM scrub typhus ELISA serology (n =16) or with CSF culture isolating bacteria known to cause bacterial meningitis (n =9). The clinical and laboratory features of the patients with scrub typhus meningitis and bacterial meningitis were compared. RESULTS: The mean age was similar in the scrub typhus and bacterial meningitis groups (44.0 ± 18.5 years vs. 46.3 ± 23.0 years). Features at admission predictive of a diagnosis of scrub typhus meningitis were duration of fever at presentation >5 days (8.4 ± 3.5 days vs. 3.3 ± 4.2 days, P < 0.001), CSF white cell count of a lesser magnitude (83.2 ± 83.0 cells/cumm vs. 690.2 + 753.8 cells/cumm, P < 0.001), CSF lymphocyte proportion >50% (83.9 ± 12.5% vs. 24.8 ± 17.5% P < 0.001), and alanine aminotransferase (ALT) elevation more than 60 IU (112.5 ± 80.6 IU vs. 35 ± 21.4 IU, P =0.02). CONCLUSION: This study suggests that clinical features, including the duration of fever and laboratory parameters such as CSF pleocytosis, CSF lymphocyte proportion >50%, and ALT values are helpful in differentiating scrub typhus from bacterial meningitis.

Role of polymerase chain reaction in the diagnosis of Trichomonas vaginalis infection in human immunodeficiency virus-infected individuals from India (South).

BACKGROUND: Trichomonas vaginalis is a protozoan parasite and an etiological agent for trichomoniasis, a sexually transmitted infection (STI). Fifty to eighty percentage of women with trichomoniasis are asymptomatic and in the absence of treatment the infection persists longer. AIM: To evaluate the role of polymerase chain reaction (PCR) in the diagnosis of trichomoniasis and also to look at the frequency of infection among human immunodeficiency virus (HIV) infected women. METHODS: A non-nested PCR was standardized to detect 102 bp size amplified product of the adhesin gene of T. vaginalis. The real time performance of this assay was performed with vaginal swab samples from 198 HIV-seropositive women who attended the infectious disease clinic and compared with wet mount and culture in Diamond's modified media. RESULTS: Among the prospectively studied 198 HIV-infected women, 1 (0.51%) was positive by wet mount, 6 (3.03%) were positive by culture and 10 (5.02%) were positive by the PCR. There was a significant observed agreement between the PCR and culture (k=0.74, Z=10.7, P<0.0000). CONCLUSION: Our study showed that the PCR assay for the amplification of adhesion gene is a highly sensitive method to screen the high risk group individuals like HIV-positive women for Trichomonas vaginalis compared to the culture. Testing algorithm should be, wet mount and if negative, test by PCR as it is rapid compared to culture which takes 7 days.

HIV reverse transcriptase: structural interpretation of drug resistant genetic variants from India.

The reverse transcriptase (RT) enzyme is the prime target of nucleoside/ nucleotide (NRTI) and non-nucleoside (NNRTI) reverse transcriptase inhibitors. Here we investigate the structural basis of effects of drug-resistance mutations in clade C RT using three-dimensional structural modeling. Apropos the expectation was for unique mechanisms in clade C based on interactions with amino acids of p66 subunit in RT molecule. 3-D structures of RT with mutations found in sequences from 2 treatment naïve, 8 failed and one reference clade C have been modeled and analyzed. Models were generated by computational mutation of available crystal structures of drug bound homologous RT. Energy minimization of the models and the structural analyses were carried out using standard methods. Mutations at positions 75,101,118,190,230,238 and 318 known to confer drug resistance were investigated. Different mutations produced different effects such as alteration of geometry of the drug-binding pocket, structural changes at the site of entry of the drug (into the active site), repositioning the template bases or by discriminating the inhibitors from their natural substrates. For the mutations analyzed, NRTI resistance was mediated mainly by the ability to discriminate between inhibitors and natural substrate, whereas, NNRTI resistance affected either the drug entry or the geometry of the active site. Our analysis suggests that different mutations result in different structural effects affecting the ability of a given drug to bind to the RT. Our studies will help in the development of newer drugs taking into account the presence of these mutations and the structural basis of drug resistance.

HIV-1 with predicted CXCR4 genotype identified in clade C from India.

BACKGROUND AND OBJECTIVE: HIV-1 uses co-receptors CCR5 and CXCR4 in addition to CD4 for viral entry into cells. CCR5 is used in the early stages of HIV-1 infection, but viruses that utilize CXCR4 for viral entry emerge in the later stages. This is not common among clade C strains, with previous data from India showing the absence of the emergence of CXCR4-using strains. Sequence analysis has demonstrated that the V3 loop plays a very important role in determining the syncytium-inducing (SI) phenotype. The V3 region of the SI variants were observed to have positively charged amino acids at positions 11 and/or 25 and also a overall higher charge. This study looked at co-receptor usage among HIV-1 strains in India from individuals who were antiretroviral therapy (ART) naïve and those not responding to ART. METHODS: Amplification and sequencing of the HIV-1 env gp120 V3 region was done on 40 ART-naïve individuals, who were selected for the study based on their CD4 counts, and eight patients who had not responded to ART. The sequences were submitted to Geno2Pheno and Web PSSM. The pol gene sequences of these strains were submitted to the REGA HIV-1 subtyping tool. RESULTS: Forty-seven strains were identified as clade C and one strain as clade A1. Geno2Pheno identified three CXCR4-using strains, and the Web PSSM clade C matrix identified two. CONCLUSION: We report, for the first time, CXCR4-using strains among HIV-1 clade C strains circulating in India.

Structural basis of drug resistance by genetic variants of HIV type 1 clade c protease from India.

Using computer modeling of three-dimensional structures and structural information available on the crystal structures of HIV-1 protease, we investigated the structural effects of mutations, in treatment-naive and treatment-exposed individuals from India and postulated mechanisms of resistance in clade C variants. A large number of models (14) have been generated by computational mutation of the available crystal structures of drug bound proteases. Localized energy minimization was carried out in and around the sites of mutation in order to optimize the geometry of interactions present. Most of the mutations result in structural differences at the flap that favors the semiopen state of the enzyme. Some of the mutations were also found to confer resistance by affecting the geometry of the active site. The E35D mutation affects the flap structure in clade B strains and E35N and E35K mutation, seen in our modeled strains, have a more profound effect. Common polymorphisms at positions 36 and 63 in clade C also affected flap structure. Apart from a few other residues Gln-58, Asn-83, Asn-88, and Gln-92 and their interactions are important for the transition from the closed to the open state. Development of protease inhibitors by structure-based design requires investigation of mechanisms operative for clade C to improve the efficacy of therapy.

A comparison of interpretation by three different HIV type 1 genotypic drug resistance algorithms using sequences from non-clade B HIV type 1 strains.

The advent of affordable ART has benefited HIV-infected individuals. Prospective studies have shown that the availability of drug resistance reports for infected individuals has allowed more effective regimens to be prescribed as compared to a control group whose physicians had no access to drug resistance reports. There is a paucity of information on the performance of genotypic algorithms on non-clade B HIV-1 strains, especially clade C. In this study the results obtained on submission of HIV-1 RT and PR sequences of non-clade B strains to the Stanford University HIV drug resistance database (SHDB) were compared to the results obtained from Geno2Pheno (G2P) and DR_Seqan (DS). For the study, we took samples from 93 treatment-naive individuals and 21 samples from 19 infected individuals showing detectable viral load while on ART. There were discrepancies in the clade identification results obtained from the SHDB and G2P databases. This feature was not available in DS. The mean observed concordance between SHDB and G2P was 85.6% while between SHDB and DC it was 37%. When the level of concordance was determined based on exposure to ART, the G2P was found to have a better level of concordance (76.8%) to SHDB as compared to SHDB versus DS (36%). We do not have phenotypic data for the strains included in this study and hence we are not in a position to assign a particular algorithm as being superior. These results also show a possible need for a subtype-specific algorithm for interpretation of HIV-1 genotypic drug resistance.

Amino acid sequence divergence of Tat protein (exon1)of subtype B and C HIV-1 strains: Does it have implications for vaccine development?

Functional genes of HIV-1 like the tat express proteins essential for viral survival and propagation. There are variations reported in levels of Tat transactivation among the different subtypes of HIV-1. This study looked at the amino acid differences in the different regions of Tat protein (exon 1) of subtype B and C strains of HIV-1 and tried to observe a molecular basis for protein function. HIV-1 sequences of subtype B (n=30) and C (n=60) strains were downloaded from HIV-1 Los Alamos data base. Among the 60 subtype C strain sequences, 30 each were from India and Africa. A HIV-1 Tat protein (exon 1) sequence, the consensus B and C sequence was obtained from the 'sequence search interface' in the Los Alamos HIV-1 sequence data. The sequences were visualized using Weblogo and the RNA binding regions of the three consensus sequences were also determined using BindN software program. Compared to subtype B, there was a high level of divergence in the auxiliary domain of tat exon 1 (amino acid positions 58- 69). The net charge of the subtype C (Indian) Tat protein (exon 1) auxiliary domain was -1.9 at pH 7 and it had an isoelectric point of 4.1. The net charge of the subtype C (African) auxiliary domain was -2.9 at pH 7 and it had an isoelectric point of 3.7 while the net charge of same region in subtype B was -0.9 at pH 7 with an isoelectric point of 4.9. The ratio of the hydrophilic residues to the total number of residues was 60% in the in both the Indian and African subtype C in the auxiliary domain while this was 50% in subtype B. The consensus subtype B sequence was found to have 36 RNA binding sites while subtype C (India) had 33 and subtype C (Africa) had 32 RNA binding sites. The HIV-1 Tat-TAR interaction is a potential target for inhibitors and being considered for its potential use in HIV-1 vaccines. Development of such inhibitor/vaccines would have to take into consideration the variation in amino acid sequence analyzed in this study as this could determine epitope presentation on MHC class I antigen for afferent immune response.