Merozoite surface protein-1 genetic diversity in Plasmodium malariae and Plasmodium brasilianum from Brazil.

BACKGROUND: The merozoite surface protein 1 (MSP1) gene encodes the major surface antigen of invasive forms of the Plasmodium erythrocytic stages and is considered a candidate vaccine antigen against malaria. Due to its polymorphisms, MSP1 is also useful for strain discrimination and consists of a good genetic marker. Sequence diversity in MSP1 has been analyzed in field isolates of three human parasites: P. falciparum, P. vivax, and P. ovale. However, the extent of variation in another human parasite, P. malariae, remains unknown. This parasite shows widespread, uneven distribution in tropical and subtropical regions throughout South America, Asia, and Africa. Interestingly, it is genetically indistinguishable from P. brasilianum, a parasite known to infect New World monkeys in Central and South America. METHODS: Specific fragments (1 to 5) covering 60 % of the MSP1 gene (mainly the putatively polymorphic regions), were amplified by PCR in isolates of P. malariae and P. brasilianum from different geographic origin and hosts. Sequencing of the PCR-amplified products or cloned PCR fragments was performed and the sequences were used to construct a phylogenetic tree by the maximum likelihood method. Data were computed to give insights into the evolutionary and phylogenetic relationships of these parasites. RESULTS: Except for fragment 4, sequences from all other fragments consisted of unpublished sequences. The most polymorphic gene region was fragment 2, and in samples where this region lacks polymorphism, all other regions are also identical. The low variability of the P. malariae msp1 sequences of these isolates and the identification of the same haplotype in those collected many years apart at different locations is compatible with a low transmission rate. We also found greater diversity among P. brasilianum isolates compared with P. malariae ones. Lastly, the sequences were segregated according to their geographic origins and hosts, showing a strong genetic and geographic structure. CONCLUSIONS: Our data show that there is a low level of sequence diversity and a possible absence of allelic dimorphism of MSP1 in these parasites as opposed to other Plasmodium species. P. brasilianum strains apparently show greater divergence in comparison to P. malariae, thus P. malariae could derive from P. brasilianum, as it has been proposed.

CRISPR/CAS as a Powerful Tool for Human Immunodeficiency Virus Cure: A Review.

Despite care and the availability of effective antiretroviral treatment, some human immunodeficiency virus (HIV)-infected individuals suffer from neurocognitive disorders associated with HIV (HAND) that significantly affect their quality of life. The different types of HAND can be divided into asymptomatic neurocognitive impairment, mild neurocognitive disorder, and the most severe form known as HIV-associated dementia. Little is known about the mechanisms of HAND, but it is thought to be related to infection of astrocytes, microglial cells, and macrophages in the human brain. The formation of a viral reservoir that lies dormant as a provirus in resting CD4+ T lymphocytes and in refuge tissues such as the brain contributes significantly to HIV eradication. In recent years, a new set of tools have emerged: the gene editing based on the clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system, which can alter genome segments by insertion, deletion, and replacement and has great therapeutic potential. This technology has been used in research to treat HIV and appears to offer hope for a possible cure for HIV infection and perhaps prevention of HAND. This approach has the potential to directly impact the quality of life of HIV-infected individuals, which is a very important topic to be known and discussed.

Single-round multiplex PCR with species-specific mitochondrial primers of P. falciparum, P. vivax/P. simium and P. malariae/P. brasilianum: Comparison with standard techniques.

A single-round multiplex PCR (mPCR) with species-specific primers (SSP) of three mitochondrial genes of Plasmodium, namely COX I, COX III and CYT B, was compared to microscopy and 18S rRNA semi-nested PCR, nested-PCR and Real Time PCRs (*PCRs). Each parasite has between 20 and 150 mitochondria and each mitochondria has one copy of each target gene, while 18S rRNA gene is repeated 4 to 8 times. The specificity of mPCR was assessed by testing Plasmodium from rodents and birds, parasites responsible for other endemic diseases in the country such as schistosomiasis, Chagas disease and leishmaniasis in addition to microorganisms that, like Plasmodium, can cause anemia (Bartonella henselae, Babesia vogeli, Rickettsia vini). No cross-reactions were detected. From a total of 149 specimens from suspected cases of malaria were tested, 97 were positive by microscopy (49 P. falciparum, 38 P. vivax, 6 P. malariae, 4 P. falciparum/P. vivax- mixed infections) and 52 were negative; 148 samples were positive by *PCRs (49 P. falciparum, 53 P. vivax, 7 P. malariae and 39 mixed infections) and one was negative; 146 were positive by mPCR (49 P. falciparum, 56 P. vivax, 9 P. malariae and 32 mixed infections) and three were negative. The comparison of groups found statistically significant differences between microscopy vs.*PCRs or vs. mPCR (p-values <0.0001), but no difference was found between mPCR vs. *PCRs (p=0.946). The agreement in the identification of Plasmodium species was only regular, with Kappa indices of 0.407 (microscopy vs. *PCRs), 0.433 (microscopy vs. mPCR) and 0.558 (*PCRs vs. mPCR). In conclusion, the diagnostic performance of mPCR was comparable to those of *PCRs, and superior to microscopy, although the identification of Plasmodium species showed many disagreements. In conclusion, a sensitive and specific one-round SSP multiplex PCR, capable of simultaneously detecting and identifying P. falciparum, P. vivax/P. simium and P. malariae/P. brasilianum may be useful in resource-constrained countries where quantitative amplifications are not yet fully accessible.

A new Real Time PCR with species-specific primers from Plasmodium malariae/P. brasilianum mitochondrial cytochrome b gene.

Plasmodium malariae mainly causes asymptomatic submicroscopic parasitemia in the endemic Amazon and non-endemic Atlantic Forest, where the number of cases and transmission of malaria through blood transfusion has increased. This study developed a P. malariae/P. brasilianum Real Time PCR (rtPCR) targeting the cytochrome b oxidase (cytb), a highly repetitive gene (20-150 copies/parasite) that should detect more cases than the 18S rRNA (4-8 copies/parasite) gene-based amplification systems. Cytb from human and non-human Plasmodium species (including P. brasilianum) aligned to the only 20 African P. malariae cytb sequences identified polymorphic regions within which we designed P. malariae species-specific primers. Non-human Plasmodium species, related parasites, anemia-causing microorganisms, normal human DNA and 47 blood bank donors samples that were truly negative to malaria accessed rtPCR specificity. Truly positive samples (n = 101) with species identification by semi-nested, nested or TaqMan PCR, and four samples from the Atlantic Forest that were suspected of malaria but three of them had negative genus TaqMan and 18S rRNA nested PCR. The cloned amplification product used in standard curves determined qPCR detection limit (0.5-1 parasite equivalent/µL). The 10 positive P. malariae samples among truly positives yielded positive rtPCR results and more importantly, rtPCR detected the four samples suspected of malaria from the Atlantic Forest. The rtPCR specificity was 100%, reproducibility 11.1% and repeatability 6.7%. In conclusion, the proposed rtPCR is fast, apparently more sensitive than all 18S rRNA amplification systems for detecting extremely low parasitemia. The rtPCR is also specific to P. malariae/P. brasilianum species. This new molecular tool could be applied to the detection of P. malariae/brasilianum infections with submicroscopic parasitemias in the context of epidemiological studies and blood bank safety programs.

PRELIMINARY REPORT ON THE PUTATIVE ASSOCIATION OF IL10 -3575 T/A GENETIC POLYMORPHISM WITH MALARIA SYMPTOMS.

Only a small percentage of individuals living in endemic areas develop severe malaria suggesting that host genetic factors may play a key role. This study has determined the frequency of single nucleotide polymorphisms (SNPs) in some pro and anti-inflammatory cytokine gene sequences: IL6 (-174; rs1800795), IL12p40 (+1188; rs3212227), IL4 (+33; rs2070874), IL10 (-3575; rs1800890) and TGFb1 (+869; rs1800470), by means of PCR-RFLP. Blood samples were collected from 104 symptomatic and 37 asymptomatic subjects. Laboratory diagnosis was assessed by the thick blood smear test and nested-PCR. No association was found between IL6 (-174), IL12p40 (+1188), IL4 (+33), IL10 (- 3575), TGFb1 (+869) SNPs and malaria symptoms. However, regarding the IL10 -3575 T/A SNP, there were significantly more AA and AT subjects, carrying the polymorphic allele A, in the symptomatic group (c2 = 4.54, p = 0.01, OR = 0.40 [95% CI - 0.17- 0.94]). When the analysis was performed by allele, the frequency of the polymorphic allele A was also significantly higher in the symptomatic group (c2 = 4.50, p = 0.01, OR = 0.45 [95% CI - 0.21-0.95]). In conclusion, this study has suggested the possibility that the IL10 - 3575 T/A SNP might be associated with the presence and maintenance of malaria symptoms in individuals living in endemic areas. Taking into account that this polymorphism is related to decreased IL10 production, a possible role of this SNP in the pathophysiology of malaria is also suggested, but replication studies with a higher number of patients and evaluation of IL10 levels are needed for confirmation.

The performance of four molecular methods for the laboratory diagnosis of congenital toxoplasmosis in amniotic fluid samples.

INTRODUCTION: Toxoplasmosis may be life-threatening in fetuses and in immune-deficient patients. Conventional laboratory diagnosis of toxoplasmosis is based on the presence of IgM and IgG anti-Toxoplasma gondii antibodies; however, molecular techniques have emerged as alternative tools due to their increased sensitivity. The aim of this study was to compare the performance of 4 PCR-based methods for the laboratory diagnosis of toxoplasmosis. One hundred pregnant women who seroconverted during pregnancy were included in the study. The definition of cases was based on a 12-month follow-up of the infants. METHODS: Amniotic fluid samples were submitted to DNA extraction and amplification by the following 4 Toxoplasma techniques performed with parasite B1 gene primers: conventional PCR, nested-PCR, multiplex-nested-PCR, and real-time PCR. Seven parameters were analyzed, sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), negative likelihood ratio (NLR) and efficiency (Ef). RESULTS: Fifty-nine of the 100 infants had toxoplasmosis; 42 (71.2%) had IgM antibodies at birth but were asymptomatic, and the remaining 17 cases had non-detectable IgM antibodies but high IgG antibody titers that were associated with retinochoroiditis in 8 (13.5%) cases, abnormal cranial ultrasound in 5 (8.5%) cases, and signs/symptoms suggestive of infection in 4 (6.8%) cases. The conventional PCR assay detected 50 cases (9 false-negatives), nested-PCR detected 58 cases (1 false-negative and 4 false-positives), multiplex-nested-PCR detected 57 cases (2 false-negatives), and real-time-PCR detected 58 cases (1 false-negative). CONCLUSIONS: The real-time PCR assay was the best-performing technique based on the parameters of Se (98.3%), Sp (100%), PPV (100%), NPV (97.6%), PLR (âˆz), NLR (0.017), and Ef (99%).

The performance of four molecular methods for the laboratory diagnosis of congenital toxoplasmosis in amniotic fluid samples

Introduction Toxoplasmosis may be life-threatening in fetuses and in immune-deficient patients. Conventional laboratory diagnosis of toxoplasmosis is based on the presence of IgM and IgG anti-Toxoplasma gondii antibodies; however, molecular techniques have emerged as alternative tools due to their increased sensitivity. The aim of this study was to compare the performance of 4 PCR-based methods for the laboratory diagnosis of toxoplasmosis. One hundred pregnant women who seroconverted during pregnancy were included in the study. The definition of cases was based on a 12-month follow-up of the infants. Methods Amniotic fluid samples were submitted to DNA extraction and amplification by the following 4 Toxoplasma techniques performed with parasite B1 gene primers: conventional PCR, nested-PCR, multiplex-nested-PCR, and real-time PCR. Seven parameters were analyzed, sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), negative likelihood ratio (NLR) and efficiency (Ef). Results Fifty-nine of the 100 infants had toxoplasmosis; 42 (71.2%) had IgM antibodies at birth but were asymptomatic, and the remaining 17 cases had non-detectable IgM antibodies but high IgG antibody titers that were associated with retinochoroiditis in 8 (13.5%) cases, abnormal cranial ultrasound in 5 (8.5%) cases, and signs/symptoms suggestive of infection in 4 (6.8%) cases. The conventional PCR assay detected 50 cases (9 false-negatives), nested-PCR detected 58 cases (1 false-negative and 4 false-positives), multiplex-nested-PCR detected 57 cases (2 false-negatives), and real-time-PCR detected 58 cases (1 false-negative). Conclusions The real-time PCR assay was the best-performing technique based on the parameters of Se (98.3%), Sp (100%), PPV (100%), NPV (97.6%), PLR (∞), NLR (0.017), and Ef (99%). .